Engineered polypeptide agents for targeted broad spectrum influenza neutralization

ABSTRACT

The present invention provides novel agents for broad spectrum influenza neutralization. The present invention provides agents for inhibiting influenza infection by bind to the influenza virus and/or hemagglutinin (HA) polypeptides and/or HA receptors, and reagents and methods relating thereto. The present invention provides a system for analyzing interactions between infolds and the interaction partners that bind to them.

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional patentapplication Ser. No. 61/298,776, filed on Jan. 27, 2010, the entiredisclosure of which is incorporated herein by reference. In accordancewith 37 CFR 1.52(e)(5), a Sequence Listing in the form of a text file(entitled “Sequence Listing.txt,” created on Jan. 26, 2011, and 49kilobytes) is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

The invention was made with government support under Grant No. R01GM057073 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Influenza virus is a global health threat that is responsible for over300,000 deaths annually. The virus evades immune recognition by engagingin a combination of accelerated antigenic drift, domain reassortment,genetic recombination, and glycosylation based masking of its surfaceglycoproteins. This rapid mutation capability of the virus isparticularly exacerbated in the context of the growing threat from thepresent H1N1 ‘swine flu’ pandemic as well as the alarming worldwidespate in recent infections with highly pathogenic avian H5N1 ‘bird flu’influenza strains. (Khanna et al., Journal of Biosciences, 33(4):475,2008, Soundararaj an et al., Nature Biotechnology 27:510, 2009).Furthermore, two of the major flu pandemics of the last centuryoriginated from avian flu viruses that changed their genetic makeup toallow for human infection.

Given the high degree of unpredictability in evolution of theseinfluenza viruses, there is a need for the development of cross-straineffective (universal or broad spectrum) anti-influenza prophylactics andtherapeutics. Such universal or broad spectrum anti-influenza agentswould augment the annual flu vaccines that are designed to targetspecific ‘seasonal’ viral strains in circulation. (Ekiert et al.,Science, 324(5924):246, 2009 and Sui et al., Nat Struct Mol Biol.16(3):265, 2009). The importance of such agents is highlighted by theemerging drug resistance to antivirals oseltamivir (TAMIFLU®)/zanamivir(RELENZA®) (NA-inhibitors) and Amantadine/Rimantadine (MP-2 inhibitors)(Collins et al., Nature 453:1258, Stouffer et al., Nature, 451:596,2008, Pielak et al., Proc. Natl. Acad. Sci. USA, 106:7379, 2009). Forinstance, over 98% and 100% of H1N1 strains this season are resistant tooseltamivir (TAMIFLU®) and the adamantane derivatives(Amantadine/Rimantadine), respectively. Additionally, seasonal fluvaccines are developed based on predictions of the most virulentinfluenza strain. In some cases, these predictions are wrong, therebymaking the seasonal flu vaccines less effective. For these reasons,there is a need for the development of broad spectrum vaccines andtherapeutic agents that are effective in the treatment or the delay ofonset of disease caused by influenza viruses, independent of subtype orclade. Of course, there is also significant value in agents that areeffective against any influenza strain, and indeed there can be profoundvalue in agents that are specific to one or a set of strains.

SUMMARY OF THE INVENTION

The present invention provides novel agents for inhibiting influenzainfection. In some embodiments, the present invention provides agentsthat bind to an influenza virus (e.g., to the virus' HA polypeptide)and/or that bind the HA receptor. In some embodiments, the presentinvention provides novel agents for broad spectrum influenzaneutralization.

In particular, the present invention provides polypeptide agents, termed“infold agents”, that bind to particular regions on an hemagglutinin(HA) polypeptide. For example, the present invention provides infoldagents that bind the membrane proximal epitope region (MPER) region ofthe HA polypeptide. In some embodiments, infold agents bind to the MPERregion independent of HA glycosylation. In some embodiments, infoldagents interact with one or more amino acid residues in the HApolypeptide, and/or with one or more glycans bound to the HApolypeptide. In some embodiments, infold agents bind N-linked glycans onthe HA polypeptide. In some embodiments, infold agents bindMPER-proximal N-glycans on the HA polypeptide.

In some embodiments, infold agents bind HA receptors. In certainembodiments, infold agents bind sialylated glycans on HA receptors. Insome embodiments, infold agents bind to sialylated glycans havingumbrella topology. In certain embodiments, infold agents bind with highaffinity and/or specificity to umbrella-topology glycans (e.g., ascompared with binding to glycans of other topologies, such ascone-topology glycans).

In some embodiments, infold agents compete with hemagglutinin forbinding to an HA receptor. In some embodiments, infold agents competewith HA for binding to umbrella-topology glycans.

In some embodiments, infold agents are characterized by a backbone foldstructure selected and dimensioned to fit within a predeterminedthree-dimensional space (e.g., binding pocket) and to display selected“interaction residues” such that they are positioned inthree-dimensional space within a designated distance from identified“target residues” in the HA polypeptide and/or HA receptor. In someembodiments, an infold agent is characterized by a first backbone foldstructure selected and dimensioned to fit within an HA polypeptidebinding site, and a second backbone fold structure selected anddimensioned to fit within an HA receptor binding site.

The present invention further provides various reagents and methodsassociated with infold agents including, for example, systems foridentifying them, strategies for preparing them, antibodies that bind tothem, and various diagnostic and therapeutic methods relating to them.Further description of certain embodiments of these aspects, and others,of the present invention, is presented below.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Presents approaches for achieving targeted and full (broad)spectrum influenza neutralization using a targeted broad spectrummolecule (Infold agent). (a) Infold agents that bind to HA MPER (redcircle) and to HA receptors (e.g., to sialylated glycans on the HAreceptors) (green circle); (b) Infold agent that bind to HA MPER (redcircle), MPER-proximal N-glycan (black circle) and sialylated glycans onHA receptors (green circle); (c) Infold agent that bind to HA MPER (redcircle); (d) Infold agent that bind to HA MPER (red circle) andMPER-proximal N-glycan (black circle).

FIG. 2. Conservation of the membrane proximal epitope region (MPER) forgroup-1 and group-2 influenza strains (Stouffer et al., Nature, 451:596,2008). The key MPER residues on HA-1 (globular head domain) and HA-2(stalk domain) are shown and the prominent amino acids from each strainin these positions are colored according to the degree of cross-cladeconservation (orange=most conserved positions).

FIG. 3. Structural rationale for limitations of antibodies forN-glycosylated influenza HA MPER targeting. F10 antibody (pink) dockedto H3 HA (wheat/gray) N-glycosylated at Asn-38 (green carbon) shows thatthe additional C′ and C″ beta-strands (black) that constitute two of thenine beta-strands in all antibody VH domains are responsible primarilyfor the steric hindrance to antibody binding.

FIG. 4. Structural rationale for reduction of MPER area available fortargeting on glycosylated HA. On non-glycosylated HA polypeptides (top),MPER area of 28*11.5˜325 A² is available for targeting, whereas areduced MPER area of 0.5*(12.3*15.3+9.5*12.2)˜152 A² is only availablefor binding on N-glycosylated HA polypeptides (bottom).

FIG. 5. Illustrates different sets of possible target residues in an HApolypeptide and specifically in and/or around the HA polypeptide MPERregion. The particular HA polypeptide depicted is an H3 HA polypeptide,from Protein Data Bank ID (PDB ID) entry 1HGG.

FIG. 6. (Top) Binding of Infold-3 to non-glycosylated HA MPER. (Bottom)Infold-3 (red) binds to the N-glycosylated HA MPER (gray/wheat) byaccommodating the N-glycan (green carbon) owing to its smaller volumeand lack of the two additional strands relative to that of antibodies asshown in FIG. 3.

FIG. 7. Presents binding of an exemplary infold agent as compared to thebinding of the C179 antibody to selected HA polypeptides, e.g., H1, H3,H5, H7 and H9.

FIG. 8. Presents an α2,6-sialylated glycan (umbrella topology) HAreceptor (cyan), shown here bound to the 2009 swine-origin influenza(commonly referred to as “pandemic influenza”) H1N1 HA polypeptide(gray).

FIG. 9. Presents an example of an α2,6 sialylated glycan recognitionmotif, used in the design of Infold-9 and Infold-10 binding to α2,6sialylated glycan HA receptors for targeted anti-influenza therapeuticdelivery.

FIG. 10. Presents framework for understanding glycan receptorspecificity. α2,3- and/or α2,6-linked glycans can adopt differenttopologies. It is believed that the ability of an HA polypeptide to bindto certain of these topologies confers upon it the ability to mediateinfection of different hosts, for example in humans. The presentinvention refers to two particularly relevant topologies, a “cone”topology (left panel) and an “umbrella” topology (right panel). The conetopology can be adopted by α2,3- and/or α2,6-linked glycans, and istypical of short oligosaccharides or branched oligosaccharides attachedto a core (although this topology can be adopted by certain longoligosaccharides). The umbrella topology can only be adopted byα2,6-linked glycans (presumably due to the increased conformationalplurality afforded by the extra C5-C6 bond that is present in the α2,6linkage), and is predominantly adopted by long oligosaccharides orbranched glycans with long oligosaccharide branches, particularlycontaining the motif Neu5Acα2,6Galβ1-3/4GlcNAc.

FIG. 11. Illustrates exemplary cone topologies. This Figure illustratescertain exemplary (but not exhaustive) glycan structures that adopt conetopologies.

FIG. 12. Illustrates exemplary umbrella topologies. (A) Certainexemplary (but not exhaustive) N- and O-linked glycan structures thatcan adopt umbrella topologies. (B) Certain exemplary (but notexhaustive) O-linked glycan structures that can adopt umbrellatopologies.

FIG. 13. Illustrates possible sets of infold agent interaction and/orbinding residues selected and designed to interact and/or bind with theindicated target residues in an HA polypeptide. As shown, the HApolypeptide is an H1 HA polypeptide.

FIG. 14. Presents images of the structure of the PDB ID 2V5Y (proteindata bank identification number) in ribbon and sticks representativemodels.

FIG. 15. Illustrates that an exemplary infold agent inhibits PR8 Virus(H1N1) influenza infectivity in vitro. The left panel highlights thatthe exemplary infold agent inhibits virus-induced plaque production in adose-dependent manner for 6 different doses.

FIG. 16. Compares the activity of an exemplary infold agent to that of acontrol (BSA) and C179 antibody using a plaque assay with 4 doses. Asshown by the plaque assay, the exemplary infold agent inhibits influenzainfectivity.

FIG. 17. Presents a H1N1 challenge in mice. In this challenge, anexemplary infold agent demonstrates delayed onset of H1N1 in mice ascompared to the PBS control. The exemplary infold agent shows a similardelay of onset to that of the antiviral drug, Ribavirin, starting ataround day five.

FIG. 18. Presents mice treated with an exemplary infold agent in an H1N1challenge. In this challenge, mice treated with the exemplary infoldagent have the least percent of weight loss post infection as comparedto the PBS control or the antiviral drug, Ribavirin.

FIG. 19. Presents phylogenetic relationships among HA subtypes, showingdivisions of influenza virus subtypes into groups, clades and clusters.

FIG. 20. Presents exemplary assessment of neutralizing activity of aparticular infold agent (Infold-28) described herein using crystalviolet.

FIG. 21. Presents exemplary assessment of Infold Agent-28 activity inMDCK using RT-PCR to directly quantify viral genome amounts. Resultsshow that the tested infold agent (Infold-28) is a potent inhibitor;IC₅₀ is influenced by the multiplicity of infection (moi).

FIG. 22. Presents effects of Infold Agent-28 pre-incubation with PR8 oninfectivity in MDCK cells. After infection, MDCK cells were grown invirus-free media either with (a) or without (b) Infold Agent-28 for 48hours before viral titer was quantified by real time PCR using primersspecific to the virus M protein. =IC₅₀ value calculated.

DEFINITIONS

Affinity: As is known in the art, “affinity” is a measure of thetightness with a particular ligand (e.g., an HA polypeptide or infoldagent) binds to its partner (e.g., and HA receptor). Affinities can bemeasured in different ways.

Amino acid: As used herein, “amino acid” refers to any natural orunnatural amino acid (see definitions of “natural amino acid” and“unnatural amino acid” below).

Antibody: As used herein, the term “antibody” is intended to includeimmunoglobulins and fragments thereof which are specifically reactive tothe designated protein or peptide, or fragments thereof. Suitableantibodies include, but are not limited to, human antibodies, primatizedantibodies, chimeric antibodies, bi-specific antibodies, humanizedantibodies, conjugated antibodies (i.e., antibodies conjugated or fusedto other proteins, radiolabels, cytotoxins), Small ModularImmunoPharmaceuticals (“SMIPs™”), single chain antibodies, cameloidantibodies, and antibody fragments. As used herein, the term“antibodies” also includes intact monoclonal antibodies, polyclonalantibodies, single domain antibodies (e.g., shark single domainantibodies (e.g., IgNAR or fragments thereof)), multispecific antibodies(e.g. bi-specific antibodies) formed from at least two intactantibodies, and antibody fragments so long as they exhibit the desiredbiological activity. Antibodies for use herein may be of any type (e.g.,IgA, IgD, IgE, IgG, IgM).

In some embodiments, an antibody is an antibody fragment. It will beappreciated that an antibody fragment may include a portion of an intactantibody, such as, for example, the antigen-binding or variable regionof an antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; triabodies; tetrabodies; linear antibodies;single-chain antibody molecules; and multi specific antibodies formedfrom antibody fragments. In some embodiments, an antibody fragment alsoincludes any synthetic or genetically engineered protein that acts likean antibody by binding to a specific antigen to form a complex. Forexample, antibody fragments may include isolated fragments, “Fv”fragments, consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy chain variable regions are connected by a peptide linker(“ScFv proteins”), and minimal recognition units consisting of the aminoacid residues that mimic the hypervariable region.

β-sandwich fold: A “β-sandwich fold” is a polypeptide domain that hasbetween 5-12β-strands when its structure is determined experimentally orpredicted computationally by any method, with a Cα RMSD (root meansquare deviation) less than or equal to 6 angstroms upon superpositiononto residues 260-355 (chain A) of the structure with the PBD ID 2V5Y(see FIG. 14). Further, such RMSD upon superposition of secondarystructural regions (excluding loops) may be less than or equal to 5 Å.Infold domains constitute the “target recognition” domains of infoldagents.

Binding: It will be understood that the term “binding”, as used herein,typically refers to a non-covalent association between or among agents.In many embodiments herein, binding is addressed with respect toparticular HA polypeptides, particular glycans (e.g., N-linked glycans,umbrella topology glycans or cone topology glycans) or particular HAreceptors. It will be appreciated by those of ordinary skill in the artthat such binding may be assessed in any of a variety of contexts. Insome embodiments, binding is assessed with respect to the HApolypeptide. In some embodiments, binding is assessed with respect toglycans attached (e.g., covalently linked to) a carrier. In some suchembodiments, the carrier is a polypeptide. In some embodiments, bindingis assessed with respect to glycans attached to an HA receptor. In suchembodiments, reference may be made to receptor binding or to glycanbinding.

Binding site: The term “binding site”, as used herein, refers to aregion of a target polypeptide, formed in three-dimensional space, thatincludes the interaction residues of the target polypeptide. As will beunderstood by those of ordinary skill in the art, a binding site mayinclude residues that are adjacent to one another on a linear chain,and/or that are distal to one another on a linear chain but near to oneanother in three-dimensional space when the target polypeptide isfolded. A binding site may comprise amino acid residues and/orsaccharide residues.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active. In particularembodiments, where a protein or polypeptide is biologically active, aportion of that protein or polypeptide that shares at least onebiological activity of the protein or polypeptide is typically referredto as a “biologically active” portion.

Broad spectrum: As used herein, the phrase “broad spectrum” refers toinfold agents that bind a variety of HA polypeptides from differentinfluenza virus strains. In some embodiments, broad spectrum infoldagents bind to a plurality of different HA polypeptides. Exemplary suchHA polypeptides include, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11,H12, H13, H14, H15, and/or H16 polypeptides, or combinations thereof. Insome embodiments, provided infold agents are broad spectrum in that theybind to HA polypeptides from at least two different clades or clustersof virus. In some embodiments provided infold agents are broad spectrumin that they bind to HA polypeptides from all known clades of virus. Insome embodiments, provided infold agents are broad spectrum in that theybind to HA polypeptides from group 1 and group 2 influenza viruses. Insome embodiments, broad spectrum refers to HA polypeptides that mediateinfection of particular hosts, e.g., avian, camel, canine, cat, civet,equine, human, leopard, mink, mouse, seal, stone martin, swine, tiger,whale, etc.

Candidate substrate: As used herein, the phrase “candidate substrate”refers to the substrates of one or more infold agents. In someembodiments, candidate substrates include but are not limited topolypeptides and saccharides. In some embodiments, candidate substratesinclude regions of HA polypeptides, the MPER region of HA-polypeptides,N-glycans on HA polypeptides, HA receptors, sialylated HA receptors,glycans on sialylated HA receptors and/or umbrella topology glycans onsialylated HA receptors.

Characteristic portion: As used herein, the phrase a “characteristicportion” of a protein or polypeptide is one that contains a continuousstretch of amino acids, or a collection of continuous stretches of aminoacids, that together are characteristic of a protein or polypeptide.Each such continuous stretch generally will contain at least two aminoacids. Furthermore, those of ordinary skill in the art will appreciatethat typically at least 5, 10, 15, 20 or more amino acids are requiredto be characteristic of a protein. In general, a characteristic portionis one that, in addition to the sequence identity specified above,shares at least one functional characteristic with the relevant intactprotein.

Characteristic sequence: A “characteristic sequence” is a sequence thatis found in all members of a family of polypeptides or nucleic acids,and therefore can be used by those of ordinary skill in the art todefine members of the family.

Combination Therapy: The term “combination therapy”, as used herein,refers to those situations in which two or more different agents areadministered in overlapping regimens so that the subject issimultaneously exposed to both agents.

Cone topology: The phrase “cone topology” is used herein to refer to a3-dimensional arrangement adopted by certain glycans and in particularby glycans on HA receptors. As illustrated in FIG. 10, the cone topologycan be adopted by α2,3 sialylated glycans or by α2,6 sialylated glycans,and is typical of short oligonucleotide chains, though some longoligonucleotides can also adopt this conformation. The cone topology ischaracterized by the glycosidic torsion angles of Neu5Acα2,3Gal linkagewhich samples three regions of minimum energy conformations given by φ(C1-C2-O-C3/C6) value of around −60, 60 or 180 and (C2-O-C3/C6-H3/C5)samples −60 to 60. FIG. 11 presents certain representative (though notexhaustive) examples of glycans that adopt a cone topology.

Direct-binding amino acids: As used herein, the phrase “direct-bindingamino acids” refers to amino acids residues that interact directly witha binding partner (e.g., one or more amino acids, glycans, etc.).Interaction residues are typically direct-binding amino acids.

Engineered: The term “engineered”, as used herein, describes apolypeptide whose amino acid sequence has been selected by man. Forexample, an engineered infold agent has an amino acid sequence that wasselected based on preferences for corresponding amino acids atparticular sites of protein-protein interactions. In some embodiments,an engineered infold sequence has an amino acid sequence that differsfrom the amino acid sequence of HA polypeptides included in the NCBIdatabase.

Hemagglutinin (HA) polypeptide: As used herein, the term “hemagglutininpolypeptide” (or “HA polypeptide”) refers to a polypeptide whose aminoacid sequence includes at least one characteristic sequence of HA. Awide variety of HA sequences from influenza isolates are known in theart; indeed, the National Center for Biotechnology Information (NCBI)maintains a database (ncbi.nlm.nih.gov/genomes/FLU/FLU) that, as of thefiling of the present application included 9796 HA sequences. Those ofordinary skill in the art, referring to this database, can readilyidentify sequences that are characteristic of HA polypeptides generally,and/or of particular HA polypeptides (e.g., H1, H2, H3, H4, H5, H6, H7,H8, H9, H10, H11, H12, H13, H14, H15, or H16 polypeptides; or of HApolypeptides that mediate infection of particular hosts, e.g., avian,camel, canine, cat, civet, environment, equine, human, leopard, mink,mouse, seal, stone martin, swine, tiger, whale, etc. For example, insome embodiments, an HA polypeptide includes one or more characteristicsequence elements found between about residues 97 and 185, 324 and 340,96 and 100, and/or 130-230 of an HA protein found in a natural isolateof an influenza virus. In some embodiments the HA polypeptide iscomprised of the HA-1 (head) and the HA-2 (stalk) domains of HA. In someembodiments, the HA polypeptide includes the characteristic sequenceelement from the membrane proximal epitope region (MPER) of HA. In someembodiments, a region of the HA polypeptide is glycosylated. In someembodiments, a region of the HA polypeptide is non-glycosylated.

In combination: The phrase “in combination”, as used herein, refers toagents that are simultaneously administered to a subject. It will beappreciated that two or more agents are considered to be administered“in combination” whenever a subject is simultaneously exposed to both(or more) of the agents. Each of the two or more agents may beadministered according to a different schedule; it is not required thatindividual doses of different agents be administered at the same time,or in the same composition. Rather, so long as both (or more) agentsremain in the subject's body, they are considered to be administered “incombination”.

Infold agent: In general, the term “infold agent” is used herein torefer to a an agent binds to a selected binding site, which agentcomprises a polypeptide. In many embodiments, an infold agent has astructure characterized by a “fold” backbone populated by interactionresidues selected and arranged so that, when the infold agent is in thevicinity of the binding site, individual interaction residues arepositioned within a preselected distance or volume of cognate targetresidues. In some embodiments, an infold agent polypeptide is anengineered or designed polypeptide. In some embodiments, infold agentsprovided herein bind a hemagglutinin (HA) polypeptide. In someembodiments, infold agents bind to an HA polypeptide in its MPER region.In some embodiments, infold agents bind to an HA polypeptide MPER regionindependent of its glycosylation. For example, in some embodiments,infold agents are designed to be of appropriate size that their bindingto an MPER region is not prevented by its glycosylation. In someembodiments, an infold agent binds to a glycosylated MPER region with anaffinity that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of its affinity foran otherwise identical non-glycosylated MPER region. In someembodiments, infold agents have volumetric sizes between 6000-120,000Å3. In some embodiments, provided infold agents have a volumetric sizethat is equal to or less than the volumetric size of an antibody. Insome embodiments, an infold agent has a total target epitope surfacearea of approximately 20×30=600 Å2. In some embodiments, the totaltarget epitope surface area of an infold agent is less than about 10 Å2,20 Å2, 30 Å2, 40 Å2, 50 Å2, 60 Å2, 70 Å2, 80 Å2, 85 Å2, 90 Å2, 95 Å2,100 Å2, 105 Å2, 110 Å2, 115 Å2, 120 Å2, 125 Å2, 130 Å2, 135 Å2, 140 Å2,145 Å2, 150 Å2, 151 Å2, 152 Å2, 153 Å2, 154 Å2, 155 Å2, 160 Å2, 165 Å2,170 Å2, 175 Å2, 180 Å2, 185 Å2, 190 Å2, 195 Å2, 200 Å2, 210 Å2, 220 Å2,230 Å2, 240 Å2, 250 Å2, 260 Å2, 270 Å2, 280 Å2, 290 Å2, 300 Å2, 310 Å2,315 Å2, 320 Å2, 325 Å2, 330 Å2 or larger. In some embodiments, totaltarget epitope surface area is less than about 200 Å2, about 175 Å2,about 150 Å2, about 125 Å2 or smaller. In many embodiments, infoldagents have a length that is less than about 1000 amino acids. In someembodiments, infold agents have a length that is less than a maximumlength of about 1000, 975, 950, 925, 900, 875, 850, 825, 800, 775, 750,725, 700, 675, 650, 625, 600, 575, 550, 525, 500, 475, 450, 425, 400,375, 350, 325, 300, 275, 250, 240, 230, 220, 210, 200, 190, 180, 170,160, 150, 140, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75,70, 65, 60, 55, 50, 45, 40, 35, 30, 25, or 20 amino acids in length. Insome embodiments, infold agents have a length that is greater than aminimum length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79 or more amino acids in length. In someembodiments, infold agents have a length between any one of such minimumlengths and any one of such maximum lengths, so long as the maximumlength is longer than the minimum length. In some particularembodiments, an infold agent has a length between about 20 and 500, orbetween 30 and 400, or between 40 and 300, or between 80 and 250 aminoacids. In some embodiments, an infold agent has a length of about 84,88, 93, 95, 98, 104, 106, 110, 111, 116, 119, 123, 124, 132, 212, 215,244, or 245. In some embodiments, infold agents are comprised of naturalamino acids. In other embodiments, infold agents comprise one or moreunnatural amino acids. In some embodiments, infold agents are comprisedof combinations of natural and unnatural amino acids. In someembodiments, an infold agent is comprised of one, two or morepolypeptide chains that are covalently (e.g., by means of a linker) ornon-covalently associated. In some embodiments, an infold agent may belinked to, or part of, a longer polypeptide chain (e.g., a completeantibody, serum albumin, or other carrier protein) so long as the infoldagent retains its three-dimensional structure and arrangement forinteraction. In some embodiments, infold agents may be appended to theN- or C-termini of another polypeptide sequence that is or is not aninfold. In some embodiments, infold agents are incorporated into thesequence of another polypeptide that is or is not an infold, therebyseparating the polypeptide sequence into two or more segments. In someembodiments, appending the infold to the N or C termini or within thesequence of another polypeptide that is or is not an infold may allowfor at least one or more of the following: a decrease in immunogenicity,increased circulation lifetime, slower in vivo degradation, incitinglocal immune response, interaction with the immune system molecules, anincrease in volume, an increase in affinity for the infold target(s), anincrease in specificity for the infold target(s), or the use of othercommonly used therapeutic/prophylactic delivery protocols. In someembodiments, appending an infold to the N or C termini or within thesequence of another polypeptide that is or is not an infold does nothave a direct effect on binding of an infold agent to a target (e.g., anHA polypeptide, the MPER region of an HA polypeptide, N-glycans on an HApolypeptide, HA receptors or sialylated glycans on HA receptors).

In some embodiments, infold agents bind to their target binding sites byinteraction with one or more target residues. In some embodiments, suchtarget residues are amino acids, saccharides, or combinations thereof.In some embodiments the present invention provides infold agents thatbind to an HA polypeptide, N-linked glycans on an HA polypeptide, an HAreceptor, sialylated glycans on an HA receptor or various combinationsthereof. In some embodiments, the present invention provides polypeptideagents comprising a first infold agent that binds to an HA polypeptideand a second infold agent that binds to the HA receptor. In some suchembodiments, the polypeptide agent comprises a single polypeptide chainthat comprises the first and second infold, optionally connected to oneanother by way of one or more linking amino acids. In some embodiments,an infold agent that binds to an HA receptor interacts with one or moreglycans on the HA receptor. In some embodiments, infold agents bindsialylated glycans. In some embodiments, infold agents bind sialylatedglycans having an umbrella-like topology. In certain embodiments, infoldagents bind to umbrella-topology glycans with high affinity and/orspecificity. In some embodiments, infold agents show a bindingpreference for umbrella-topology glycans as compared with glycans ofother topologies (e.g., cone-topology glycans). In some embodiments,infold agents compete with HA for binding to HA receptors. In someembodiments, infold agents compete with HA for binding such that bindingbetween the HA polypeptide and the HA receptor is reduced at least 1.5fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17fold, at least 18 fold, at least 19 fold, or at least 20 fold. In someembodiments, infold agents compete with HA for binding to glycans on HAreceptors. In some embodiments, infold agents compete with HA forbinding to umbrella-topology glycans. In some embodiments, an infoldagent provided herein is an umbrella topology blocking agent. In someembodiments, an infold agent provided herein is an umbrella topologyspecific blocking agent. In some embodiments, an infold agent has abackbone fold structure populated by a plurality of direct binding aminoacid residues (i.e., amino acid residues that make direct contacts withHA amino acids or glycans), and/or with HA receptor amino acids orglycan as described herein.

Interaction residues: The term “interaction residues”, as used herein,refers to residues in an infold agent that are designed to interact withparticular target residues in a target polypeptide. Specifically,interaction residues are selected and arranged within an infold agentsequence so that they will be displayed in three dimensional spacewithin a predetermined distance (or volume) of identified targetresidues (e.g., upon binding, docking or other interaction assays). Inmany embodiments, interaction residues are direct-binding residues.

Isolated: The term “isolated”, as used herein, refers to an agent orentity that has either (i) been separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature or in an experimental setting); or (ii) produced by the handof man. Isolated agents or entities may be separated from at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the othercomponents with which they were initially associated. In someembodiments, isolated agents are more than 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% pure.

Long oligosaccharide: For purposes of the present disclosure, anoligosaccharide is typically considered to be “long” if it includes atleast one linear chain that has at least four saccharide residues.

Natural amino acid: As used herein, the term “natural amino acid” refersto one of the naturally occurring twenty amino acids. Refer to Table 1for a list of these amino acids.

Polypeptide: As used herein, a “polypeptide”, generally speaking, is astring of at least two amino acids attached to one another by a peptidebond. In some embodiments, a polypeptide may include at least 3-5 aminoacids, each of which is attached to others by way of at least onepeptide bond. Those of ordinary skill in the art will appreciate thatpolypeptides sometimes include “non-natural” amino acids or otherentities that nonetheless are capable of integrating into a polypeptidechain, optionally.

Pure: As used herein, an agent or entity is “pure” if it issubstantially free of other components. For example, a preparation thatcontains more than about 90% of a particular agent or entity istypically considered to be a pure preparation. In some embodiments, anagent or entity is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%< or99% pure.

Short oligosaccharide: For purposes of the present disclosure, anoligosaccharide is typically considered to be “short” if it has fewerthan 4, or certainly fewer than 3, residues in any linear chain.

Specificity: As is known in the art, “specificity” is a measure of theability of a particular ligand (e.g., an infold agent) to distinguishits binding partner (e.g., a human HA receptor, and particularly a humanupper respiratory tract HA receptor) from other potential bindingpartners (e.g., an avian HA receptor).

Substantial homology: The phrase “substantial homology” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially homologous” ifthey contain homologous residues in corresponding positions. Homologousresidues may be identical residues. Alternatively, homologous residuesmay be non-identical residues will appropriately similar structuraland/or functional characteristics. For example, as is well known bythose of ordinary skill in the art, certain amino acids are typicallyclassified as “hydrophobic” or “hydrophilic” amino acids., and/or ashaving “polar” or “non-polar” side chains Substitution of one amino acidfor another of the same type may often be considered a “homologous”substitution. Typical amino acid categorizations are summarized below inTable 1:

TABLE 1 Amino Acid Categorizations Amino Acid Abbreviations PolarityCharge Alanine Ala A Nonpolar neutral 1.8 Arginine Arg R Polar positive−4.5 Asparagine Asn N Polar neutral −3.5 Aspartic acid Asp D Polarnegative −3.5 Cysteine Cys C Nonpolar neutral 2.5 Glutamic acid Glu EPolar negative −3.5 Glutamine Gln Q Polar neutral −3.5 Glycine Gly GNonpolar neutral −0.4 Histidine His H Polar positive −3.2 Isoleucine IleI Nonpolar neutral 4.5 Leucine Leu L Nonpolar neutral 3.8 Lysine Lys KPolar positive −3.9 Methionine Met M Nonpolar neutral 1.9 PhenylalaninePhe F Nonpolar neutral 2.8 Proline Pro P Nonpolar neutral −1.6 SerineSer S Polar neutral −0.8 Threonine Thr T Polar neutral −0.7 TryptophanTrp W Nonpolar neutral −0.9 Tyrosine Tyr Y Polar neutral −1.3 Valine ValV Nonpolar neutral 4.2

TABLE 2 Ambiguous Amino Acids 3-Letter 1-Letter Asparagine or asparticacid Asx B Glutamine or glutamic acid Glx Z Leucine or Isoleucine Xle JUnspecified or unknown amino acid Xaa X

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul, et al.,Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990;Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.,Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999.In addition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues arehomologous over a relevant stretch of residues. In some embodiments, therelevant stretch is a complete sequence. In some embodiments, therelevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500 or more residues.

Substantial identity: The phrase “substantial identity” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially identical” ifthey contain identical residues in corresponding positions. As is wellknown in this art, amino acid or nucleic acid sequences may be comparedusing any of a variety of algorithms, including those available incommercial computer programs such as BLASTN for nucleotide sequences andBLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplarysuch programs are described in Altschul, et al., Basic local alignmentsearch tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al.,Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402,1997; Baxevanis et al., Bioinformatics: A Practical Guide to theAnalysis of Genes and Proteins, Wiley, 1998; and Misener, et al.,(eds.), Bioinformatics Methods and Protocols (Methods in MolecularBiology, Vol. 132), Humana Press, 1999. In addition to identifyingidentical sequences, the programs mentioned above typically provide anindication of the degree of identity. In some embodiments, two sequencesare considered to be substantially identical if at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more of their corresponding residues are identical over arelevant stretch of residues. In some embodiments, the relevant stretchis a complete sequence. In some embodiments, the relevant stretch is atleast 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500 or more residues.

Target polypeptide: A “target polypeptide”, as that term is used herein,is a polypeptide with which an infold agent interacts. In someembodiments, a target polypeptide is an HA polypeptide. In someembodiments, a target polypeptide is an HA receptor polypeptide.

Target residue: A “target residue”, as that term is used herein, is aresidue within a target polypeptide with which an infold agent isdesigned to interact. Specifically, an infold agent is typicallycharacterized by particular interaction residues selected and arranged(by virtue of being presented on the selected “fold” backbone) to bewithin a certain predetermined distance (or volume) of a target residue.In some embodiments, a target residue is or comprises an amino acidresidue. In some embodiments, a target residue is or comprises asaccharide residue.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that elicits a desired biological or pharmacologicaleffect.

Treatment: As used herein, the term “treatment” refers to any methodused to alleviate, delay onset, reduce severity or incidence, or yieldprophylaxis of one or more symptoms or aspects of a disease, disorder,or condition. For the purposes of the present invention, treatment canbe administered before, during, and/or after the onset of symptoms.

Umbrella topology: The phrase “umbrella topology” is used herein torefer to a 3-dimensional arrangement adopted by certain glycans and inparticular by glycans on HA receptors. The present invention encompassesthe recognition that binding to umbrella topology glycans ischaracteristic of HA polypeptides that mediate infection of human hosts.As illustrated in FIGS. 10 and 12, the umbrella topology is typicallyadopted only by α2,6 sialylated glycans, and is typical of long (e.g.,greater than tetrasaccharide) oligosaccharides. In some embodiments,umbrella-topology glycans are glycans exhibiting a three-dimensionalstructure substantially similar to the structure presented in FIG. 10(right panel). In some embodiments, umbrella-topology glycans areglycans which contact HA polypeptides via the interactions with specificamino acid residues. In some embodiments, glycan structural topology isclassified based on parameter θ defined as angle between C₂ of Sia, C₁of Gal, and C₁ of GlcNAc. Values of θ<100° represent cone-like topologyadopted by α2,3 and short α2,6 glycans. Values of θ>110° representumbrella-like topology, such as topology adopted by long α2,6 glycans.An example of umbrella topology is given by φ angle of Neu5Acα2,6Gallinkage of around −60. FIG. 12 presents certain representative (thoughnot exhaustive) examples of glycans that can adopt an umbrella topology.The long α2,6 motifs presented in FIG. 12 includes Neu5Acα2,6 linked atthe non-reducing end to a long chain (e.g., at least a trisaccharide)found as a part of biological N-linked glycans, O-linked glycans, andglycolipids. The boxed inset shows examples of the umbrella-topologylong α2,6 glycan moieties that are found as a part of biological glycansthat bind to high affinity with HA. In some embodiments,umbrella-topology glycans (e.g., at a site) comprise a greaterproportion of long (e.g. multiple lactosamine units) α2,6oligosaccharide branches than short α2,6 (e.g. single lactosamine)branches. In some embodiments, umbrella-topology glycans (e.g., at asite) comprise about 2-fold, about 3-fold, about 4-fold, about 5-fold,about 10-fold, about 20-fold, about 50-fold, or greater than about50-fold more long α2,6 oligosaccharide branches than short α2,6 (e.g.single lactosamine) branches. In certain embodiments, the uniquecharacteristic of HA interactions with umbrella-topology glycans and/orglycan decoys is the HA contact with a glycan comprising sialic acid(SA) and/or SA analogs at the non-reducing end. In some embodiments,chain length of the oligosaccharide is at least a trisaccharide(excluding the SA or SA analog). In certain embodiments, umbrellatopology glycans are oligosaccharides of the following form:

Neu5Acα2,6Sug1-Sug2-Sug3

where:

(a) Neu5Ac α2,6 is typically (but not essentially) at the non-reducingend;

(b) Sug1:

(i) is a hexose (frequently Gal or Glc) or hexosamine (GlcNAc or GalNAc)in α or β configuration (frequently β- for N- and O-linked extension andα- in the case of GalNAcα- that is O-linked to glycoprotein);(ii) no sugars other than Neu5Acα2,6 are attached to any of thenon-reducing positions of Sug1 (except when Sug1 is GalNAcα- that isO-linked to the glycoprotein); and/or(iii) non-sugar moieties such as sulfate, phosphate, guanidium, amine,N-acetyl, etc. can be attached to non-reducing positions (typically 6position) of Sug1 (e.g., to improve contacts with HA);(c) Sug2 and/or Sug3 is/are:(i) hexose (frequently Gal or Glc) or hexosamine (GlcNAc or GalNAc) in aor (3 configuration (frequently (3); and/or(ii) sugars (such as Fuc) or non-sugar moieties such as sulfate,phosphate, guanidium, amine, N-acetyl, etc. can be attached tonon-reducing positions of Sug2, Sug3, and/or Sug4;(d) Linkage between any two sugars in the oligosaccharide apart fromNeu5Acα2,6 linkage can be 1-2, 1-3, 1-4, and/or 1-6 (typically 1-3 or1-4); and/or(e) Structure where Neu5Acα2,6 is linked GalNAcα that is O-linked to theglycoprotein and additional sugars are linked to the non-reducing end ofGalNAcα for example

(i) Neu5Acα2,6(Neu5Acα2,3Galβ1-3)GalNAcα- (ii)Neu5Acα2,6(Galβ1-3)GalNAcα-

Unnatural amino acid: As used herein, the term “unnatural amino acid”refers to any amino acid, modified amino acid, and/or amino acidanalogue that is not one of the 20 naturally occurring amino acids.Refer to U.S. Pat. No. 7,045,337, U.S. Pat. No. 7,385,028, and U.S. Pat.No. 7,332,571, the entire disclosures of which are incorporated hereinby reference. As used herein, “unnatural amino acid” also encompasseschemically modified amino acids, including but not limited to salts,amino acid derivatives (such as amides), and/or substitutions. Aminoacids, including carboxy- and/or amino-terminal amino acids in peptides,can be modified by peglyation, methylation, amidation, acetylation,and/or substitution with other chemical groups that do not adverselyaffect the activity of the infold agent. Amino acids may participate ina disulfide bond. The term “amino acid” is used interchangeably with“amino acid residue,” and may refer to a free amino acid and/or to anamino acid residue of a peptide. It will be apparent from the context inwhich the term is used whether it refers to a free amino acid or aresidue of a peptide.

Universal anti-influenza agent: As used herein, the term “universalanti-influenza agent” refers to an agent that has broad-spectrumneutralization across influenza virus strains, groups, clades, andclusters (see definitions of “broad spectrum” above and FIG. 19).

Vaccination: As used herein, the term “vaccination” refers to theadministration of a composition intended to generate an immune response,for example to a disease-causing agent. For the purposes of the presentinvention, vaccination can be administered before, during, and/or afterexposure to a disease-causing agent, and in certain embodiments, before,during, and/or shortly after exposure to the agent. In some embodiments,vaccination includes multiple administrations, appropriately spaced intime, of a vaccinating composition.

Variant: As used herein, the term “variant” is a relative term thatdescribes the relationship between a particular polypeptide (e.g., HApolypeptide) of interest and a “parent” polypeptide to which itssequence is being compared. A polypeptide of interest is considered tobe a “variant” of a parent polypeptide if the polypeptide of interesthas an amino acid sequence that is identical to that of the parent butfor a small number of sequence alterations at particular positions.Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% ofthe residues in the variant are substituted as compared with the parent.In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1substituted residue as compared with a parent. Often, a variant has avery small number (e.g., fewer than 5, 4, 3, 2, or 1) number ofsubstituted functional residues (i.e., residues that participate in aparticular biological activity). Furthermore, a variant typically hasnot more than 5, 4, 3, 2, or 1 additions or deletions, and often has noadditions or deletions, as compared with the parent. Moreover, anyadditions or deletions are typically fewer than about 25, 20, 19, 18,17, 16, 15, 14, 13, 10, 9, 8, 7, 6, and commonly are fewer than about 5,4, 3, or 2 residues. In some embodiments, the parent polypeptide is onefound in nature. For example, a parent HA polypeptide may be one foundin a natural (e.g., wild type) isolate of an influenza virus (e.g., awild type HA polypeptide).

Vector: As used herein, “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. In some embodiment, vectors are capable of extra-chromosomalreplication and/or expression of nucleic acids to which they are linkedin a host cell such as a eukaryotic or prokaryotic cell. Vectors capableof directing the expression of operatively linked genes are referred toherein as “expression vectors.”

Wild type: As is understood in the art, the phrase “wild type” generallyrefers to a normal form of a protein or nucleic acid, as is found innature. For example, wild type HA polypeptides are found in naturalisolates of influenza virus. A variety of different wild type HAsequences can be found in the NCBI influenza virus sequence database,ncbi.nlm.nih.gov/genomes/FLU/FLU.

DETAILED DESCRIPTION OF CERTAIN PARTICULAR EMBODIMENTS OF THE INVENTIONHemagglutinin (HA) Polypeptide

Influenza viruses are RNA viruses which are characterized by a lipidmembrane envelope containing two glycoproteins, a hemagglutinin (HA)polypeptide and a neuraminidase (NA) polypeptide, embedded in themembrane of the virus particular. There are 16 known HA polypeptidesubtypes (H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15, and H16) and 9 NA polypeptide subtypes (N1, N2, N3, N4, N5, N6, N7,N8, and N9), and different influenza strains are named based on thenumber of the strain's HA polypeptide and NA polypeptide subtypes,wherein there are different combinations of one HA polypeptide subtypecombined with one NA polypeptide subtype (for example, H1N1, H1N2, H1N3,H1N4, H1N5, etc.).

Based on comparisons of amino acid sequence identity and of crystalstructures, the HA polypeptide subtypes have been divided into two maingroups and four smaller clades, which is further divided into fiveclusters (FIG. 19). The different HA polypeptide subtypes do notnecessarily share strong amino acid sequence identity, but the overall3D structures of the different HA polypeptide subtypes are similar toone another, with several subtle differences that can be used forclassification purposes. For example, the particular orientation of themembrane-distal subdomains in relation to a central α-helix is onestructural characteristic commonly used to determine HA polypeptidesubtype (Russell et al., Virology, 325:287, 2004).

Mature HA polypeptides are comprised of two domains, (1) a core HA-1domain known as the sialic acid-binding domain, and (2) thetransmembrane stalk of HA, known as HA-2 domain. HA-1 contains thebinding site for glycans and it is thought that HA-1 is responsible formediating binding of HA to the HA-receptor. HA-2 is responsible forpresenting the HA-1 domain.

Without wishing to be bound by any particular theory, analysis of HAsequences from all influenza subtypes showed a set of amino acids in theinterface of the HA-1 (head) and HA-2 (stalk) domains that are wellconserved and accessible to prospective therapeutic molecules (FIG. 13).Studies have also observed the excellent broad spectrum conservation ofthe HA-1/HA-2 interface membrane proximal epitope region (MPER) thatincludes the canonical α-helix and residues in its vicinity (Ekiert etal., Science, 324(5924):246, 2009; Sui et al., Nat Struct Mol Biol.16(3):265, 2009) (FIG. 2).

Antibodies including CR6261 FAb (Ekiert et al., Science, 324(5924):246,2009), and F10 scFv (Sui et al., Nat Struct Mol Biol. 16(3):265, 2009)have been developed against the highly conserved membrane proximalepitope region (MPER) of the HA (FIG. 3). While these antibodies aresuccessful in neutralization of the group-1 clade of the influenza-Astrains (H1, H2, H5, H9), they are ineffective against the group-2strains that are N-glycosylated in the proximity of their MPER (Ekiertet al., Science, 324(5924):246, 2009; Sui et al., Nat Struct Mol Biol.16(3):265, 2009). These group-2 strains include influenza-A H3, H7, andH10 that are glycosylated in Asn-38 of the N-terminal HA-1 domain, aswell as the entire influenza-B clade of viruses that are glycosylated inAsn-238 of the C-terminal HA-1 domain. Without wishing to be bound bytheory, we propose that the N-glycosylations prevent the largeantibody-based molecules such as IgGs, mAbs, and scFvs from accessingthe underlying epitope, thereby limiting their broad spectrumapplication (FIG. 4).

HA Receptors

HA polypeptides interact with the surface of cells by binding to aglycoprotein receptor, known as the HA receptor. Binding of an HApolypeptide to an HA receptor is predominantly mediated by N-linkedglycans on the HA receptors. Specifically, HA polypeptides on thesurface of flu virus particles recognizes sialylated glycans that areassociated with HA receptors on the surface of the cellular host. Afterrecognition and binding, the host cell engulfs the viral cell and thevirus is able to replicate and produce many more virus particles to bedistributed to neighboring cells.

HA polypeptides exists in the viral membrane as a homotrimer of one of16 subtypes, termed H1-H16. Only three of these subtypes (H1, H2, andH3) have thus far become adapted for human infection. One reportedcharacteristic of HA polypeptides that have adapted to infect humans(e.g., of HA polypeptides from the pandemic H1N1 (1918) and H3N2(1967-68) influenza subtypes) is their ability to preferentially bind toα2,6 sialylated glycans in comparison with their avian progenitors thatpreferentially bind to α2,3 sialylated glycans (Skehel & Wiley, Annu RevBiochem, 69:531, 2000; Rogers, & Paulson, Virology, 127:361, 1983;Rogers et al., Nature, 304:76, 1983; Sauter et al., Biochemistry,31:9609, 1992; Connor et al., Virology, 205:17, 1994; Tumpey et al.,Science, 310:77, 2005). Without wishing to be bound by any particulartheory, it has been proposed that the ability to infect human hostscorrelates less with binding to glycans of a particular linkage, andmore with binding to glycans of a particular topology. We havespecifically demonstrated that HA polypeptides that mediate infection ofhumans bind to umbrella topology glycans, often showing preference forumbrella topology glycans over cone topology glycans (even thoughcone-topology glycans may be α2,6 sialylated glycans) (See, for example,U.S. Ser. No. 12/348,266 filed Jan. 2, 2009, U.S. Ser. No. 12/301,126,filed Nov. 17, 2008, U.S. Ser. No. 61/018,783, filed Jan. 3, 2008, U.S.Ser. No. 11/969,040, filed Jan. 3, 2008, U.S. Ser. No. 11/893,171, filedAug. 14, 2007, U.S. Ser. No. 60/837,868, filed on Aug. 14, 2006, U.S.Ser. No. 60/837,869, filed on August 14, and to PCT applicationPCT/US07/18160, filed Aug. 14, 2007, each of which is incorporatedherein by reference).

Several crystal structures of HA polypeptides from H1 (human and swine),H3 (avian) and H5 (avian) subtypes bound to sialylated oligosaccharides(of both α2,3 and α2,6 linkages) are available and provide molecularinsights into the specific amino acids that are involved in distinctinteractions of the HA polypeptides with these glycans (Eisen et al.,Virology, 232:19, 1997; Ha et al., Proc Natl Acad Sci USA, 98:11181,2001; Ha et al., Virology, 309:209, 2003; Gamblin et al., Science,303:1838, 2004; Stevens et al., Science, 303:1866, 2004; Russell et al.,Glycoconj J 23:85, 2006; Stevens et al., Science, 312:404, 2006). Somecrystal structures of exemplary HA-glycan interactions have beenidentified and are presented in Table 3:

TABLE 3 Crystal Structures of HA-Glycan Complexes Glycan (with assignedAbbreviation (PDB ID) Virus Strain coordinates) ASI30_H1_23 (1RV0)A/Swine/Iowa/30 (H1N1) Neu5Ac ASI30_H1_26 (1RVT) A/Swine/Iowa/30 (H1N1)Neu5Acα6Galβ4GlcNAcβ3Galβ4Glc APR34_H1_23 (1RVX) A/Puerto Rico/8/34(H1N1) Neu5Acα3Galβ4GlcNAc APR34_H1_26 (1RVZ) A/Puerto Rico/8/34 (H1N1)Neu5Acα6Galβ4GlcNAc ADU63_H3_23 (1MQM) A/Duck/Ukraine/1/63 (H3N8)Neu5Acα3Gal ADU63_H3_26 (1MQN) A/Duck/Ukraine/1/63 (H3N8) Neu5Acα6GalAAI68_H3_23 (1HGG) A/Aichi/2/68 (H3N2) Neu5Acα3Galβ4Glc ADS97_H5_23(1JSN) A/Duck/Singapore/3/97 (H5N3) Neu5Acα3Galβ3GlcNAcADS97_H5_26(1JSO) A/Duck/Singapore/3/97 (H5N3) Neu5Ac Viet04_H5 (2FK0)A/Vietnam/1203/2004 (H5N1) HA - α2,6 sialylated glycan complexes weregenerated by superimposition of the CA trace of the HA-1 subunit ofADU63_H3 and ADS97_H5 and Viet04_H5 on ASI30_H1_26 and APR34_H1_26 (H1).Although the structural complexes of the human A/Aichi/2/68 (H3N2) withα2,6 sialylated glycans are published (Eisen et al., 1997, Virology,232: 19), their coordinates were not available in the Protein Data Bank.The SARF2 (123d.ncifcrf.gov/sarf2) program was used to obtain thestructural alignment of the different HA-1 subunits for superimposition.

For example, the crystal structures of H5 (A/duck/Singapore/3/97) aloneor bound to an α2,3 or an α2,6 sialylated oligosaccharide identifiescertain amino acids that interact directly with bound glycans, and alsoamino acids that are one or more degree of separation removed (Stevenset al., Proc Natl Acad Sci USA 98:11181, 2001). In some cases,conformation of these residues is different in bound versus unboundstates. For instance, Glu190, Lys193 and Gln226 all participate indirect-binding interactions and have different conformations in thebound versus the unbound state. The conformation of Asn186, which isproximal to Glu190, is also significantly different in the bound versusthe unbound state.

Without wishing to be bound by any particular theory, it is thought thatthe HA receptors are modified by either α2,3 or α2,6 sialylated glycansnear the receptor's HA polypeptide-binding site, and the type of linkageof the receptor-bound glycan can affect the conformation of thereceptor's HA polypeptide-binding site, thus affecting the receptor'sspecificity for different HA polypeptides. For example, the glycanbinding pocket of avian HA receptor is narrow. Without wishing to bebound by any particular theory, it has been proposed that this pocketbinds to the trans conformation of α2,3 sialylated glycans, and/or tocone-topology glycans, whether α2,3 or α2,6 linked (FIGS. 10-12).

HA receptors in avian tissues, and also in human deep lung andgastrointestinal (GI) tract tissues are characterized by α2,3 sialylatedglycan linkages, and furthermore are characterized by glycans, includingα2,3 sialylated and/or α2,6 sialylated glycans, which predominantlyadopt cone topologies. HA receptors having such cone-topology glycansmay be referred to herein as CTHArs.

By contrast, human HA receptors in the bronchus and trachea of the upperrespiratory tract are modified by α2,6 sialylated glycans. Unlike theα2,3 motif, the α2,6 motif has an additional degree of conformationalfreedom due to the C6-05 bond (Russell et al., Glycoconj J23:85, 2006).HA polypeptides that bind to such α2,6 sialylated glycans have a moreopen binding pocket to accommodate the diversity of structures arisingfrom this conformational freedom. Moreover, according to the presentinvention, HA polypeptides may need to bind to glycans (e.g., α2,6sialylated glycans) in an umbrella topology, and particularly may needto bind to such umbrella topology glycans with strong affinity and/orspecificity, in order to effectively mediate infection of human upperrespiratory tract tissues. HA receptors having umbrella-topology glycansmay be referred to herein as UTHArs.

As a result of these spatially restricted glycosylation profiles, humansare not usually infected by viruses containing many wild type avian HApolypeptides (e.g., avian H5). Specifically, because the portions of thehuman respiratory tract that are most likely to encounter virus (i.e.,the trachea and bronchi) lack receptors with cone glycans (e.g., α2,3sialylated glycans, and/or short glycans) and wild type avian HApolypeptides typically bind primarily or exclusively to receptorsassociated with cone glycans (e.g., α2,3 sialylated glycans, and/orshort glycans), humans rarely become infected with avian viruses. Onlywhen in sufficiently close contact with virus that it can access thedeep lung and/or gastrointestinal tract receptors having umbrellaglycans (e.g., long α2,6 sialylated glycans) do humans become infected.

Infold Agents Infold Agent Structure

As described herein, infold agents are, generally, polypeptide agentsthat bind to a selected binding site. In many embodiments, an infoldagent has a structure characterized by a “fold” backbone populated byinteraction residues selected and arranged so that, when the infoldagent is in the vicinity of the binding site, individual interactionresidues are positioned within a preselected distance or volume ofcognate target residues.

A variety of polypeptide “fold” structures, appropriately utilized asinfold agent backbone structures according to the present invention, areknown in the art. That is, it is well known that linear chains of aminoacids adopt discrete secondary and tertiary structures, so that aminoacids that are proximal in space, but distal in sequence. For example,common secondary folds include α-helix, β-sheet, polyproline helix, 3₁₀helix and turns. Common tertiary folds include ferredoxin-like (4.45%),TIM-barrel (3.94%), P-loop containing nucleotide triphosphate hydrolase(3.71%), protein kinases (PK) catalytic domain (3.14%), NAD(P)-bindingRossmann-fold domains (2.80%), DNA:RNA-binding 3-helical bundle (2.60%),α-α superhelix (1.95%), S-adenosyl-L-methionine-dependentmethyltransferase (1.92%), 7-bladed beta-propeller (1.85%),α:β-hydrolases (1.84%), PLP-dependent transferase (1.61%), adeninenucleotide α-hydrolase (1.59%), flavodoxin-like (1.49%),immunoglobulin-like β-sandwich (1.38%), and glucocorticoid receptor-like(0.97%), where the values in parentheses are the percentages ofannotated proteins adopting the respective folds (see, for example,Zhang et al., Cellular and Molecular Life Sciences, 58:72, 2001).

In some embodiments, infold agents described herein have a fold backbonestructure based on a protein selected from the group consisting of: theextracellular region of human tissue factor, tenascin, neuroglian,neural cell adhesion molecule 1 (NCAM), integrin beta-4 subunit, growthhormone receptor, erythropoietin (EPO) receptor, prolactin receptor,inerleukin-4 receptor alpha chain, beta-chain of GM-CSF receptors,beta-chain of IL-3 receptors, beta-chain of IL-5 receptors, granulocytecolony-stimulating factor (GC-SF) receptor, contactin 3 (KIAA1496),brother of CDO precursor (BOC), interferon-gamma receptor alpha chain,cytokine receptor gp130 cytokine-binding domains, interleukin-10receptor 1 (IL-10R1), type 1 titin module, the p40 domain ofinterleukin-12 (IL-12 beta chain), interleukin-6 receptor alpha chain,interferon-alpha/beta receptor beta chain, KIAA1568 protein, KIAA0343protein, KIAA1355 protein, ciliary neurotrophic factor receptor alpha,host cell factor 2 (HCF-2), ankyrin repeat domains 1 protein (FANK1),ephrin type B receptor 1, ephrin type A receptor 8, cytokine receptorcommon gamma chain, rim binding protein 2, interleukin-2 receptor betachain, tenascin-X, receptor-type tyrosine-protein phosphatase delta(PTPRD), sidekick 2, neogenin down syndrome cell adhesion molecule-likeprotein 1 (DSCAML1), mysoin binding protein C (fast-type), receptor-typetyrosine-protein phosphatase F (PTPRF), hedgehog receptor iHog, ephrintype A receptor 1.

In some embodiments, infold agents described herein have an antibodyfold backbone. Several infold agents exemplified herein (see, forexample, Table 9, e.g., Infold-23 through Infold-40) have an antibodyfold backbone. In some embodiments, infold agents described herein havea β-sandwich domain fold backbone.

In some embodiments, inventive infold agents are characterized by thepresence of interaction residues selected and arranged to interact withparticular target residues in an HA polypeptide and/or an HA receptor.For Example, Tables 4 and 5 present certain representative targetresidue sets for HA polypeptides (glycosylated or nonglycosylated; Table4) and for HA receptor (specifically HA receptor containing sialylatedglycans; Table 5) also see FIGS. 5 and 9.

TABLE 4 HA Polypeptide Exemplary Target Residue Sets (exemplified withH3 HA PBD ID 1HGG numbering) Set-T1 Trp-21, Ile-48, Ile-45, Met-320Set-T2 Val-20, Leu-38 Set-T3 Thr-37, Thr-41 Set-T4 His-18 Set-T5Lys-121, Lys-39 Set-T6 Asp-19, Gln-42, Asp-46, Gln-47, Asn-49, Asn-53,Asn-38 Set-T7 Thr-318, Thr-40 Set-T8 Leu-52, Leu-42, Ile-56, Pro-293Set-T9 His-56 Set-T10 Lys-58, Lys-292 Set-T11 Asn-290, Asp-291, Glu-57,Glu-61, Glu-280 Set-T12 Ser-279, Thr-59 HA Polypeptide Glycan ExemplaryTarget Residue Sets Set-T14 N-glycan on HA polypeptides. In someembodiments, N-glycans on HA polypeptides are near or proximal to theMPER region

TABLE 5 HA Receptor Exemplary Target Residue Sets Set-T13 Sialic acid onHA receptor glycans

In some embodiments, the present invention provides infold agents thatcontain interaction residues that bind to these target sets.

For example, Table 6 provides infold interaction residue sets that canbe utilized in inventive infold agents designed to interact with HApolypeptides and/or with HA receptors according to the rules set forthin Table 7:

TABLE 6 Exemplary Infold Interaction Residue Sets Set-In1 Ile, Leu, Val,Phe, Met, Trp, Tyr, Pro, His Set-In2 Val, Phe, Trp, Tyr, Asp, Arg, LysSet-In3 Ile, Leu, Phe, Met, Trp, Tyr, His, Gln, Asp, Arg Set-In4 Asp,Glu, Phe, Met, Tyr, Trp Set-In5 Arg, Lys, His, Asn, Gln, Thr Set-In6Tyr, Trp, Phe, His, Arg, Gln, Ser Set-In7 Tyr, Trp, Phe, Pro, Arg, Asp,His, Lys

TABLE 7 Exemplary Infold Structures Infold Interaction ResiduesPresented within 5 Å of Target Residues Target Residue Set DuringBinding or Upon Docking Set-T1 Set-In1 Set-T2 Set-In1 Set-T3 Set-In2Set-T4 Set-In3 Set-T5 Set-In4 Set-T6 Set-In5 Set-T7 Set-In2 Set-T8Set-In1 Set-T9 Set-In3 Set-T10 Set-In4 Set-T11 Set-In5 Set-T12 Set-In2Set-T13 Set-In6 Set-T14 Set-In7

Alternatively or additionally, infold agents are polypeptidescharacterized by one or more structural features set forth in Tables 4-8and/or FIGS. 5, 13, and/or 14.

For example, in some embodiments, infold agents provided herein containone or more of the interaction residue sequence elements defined in eachbox of Table 8. Each box defines one sequence element, wherein the aminoacids listed in each box are interaction residues that are eitheradjacent to one another or separated by one or two amino acids in theinfold agent polypeptide chain.

TABLE 8 Defined interaction residues for binding HA polypeptide MPER andSialylated Glycans Target Interaction Residues in Infold Agents HAPolypeptide MPER (I/V)& (M/C) (F/Y)& (D/E) & W (S/T)& (I/V/L)& (M/C)(M/C) N (F/Y)&N (M/C) (F/Y)& W&N (F/Y)&W and N (I/V/L) (S/T)& (D/E)&(D/E)& (D/E)& (S/T) &W (I/V/L) (I/V/L) (M/C) (F/Y) (I/V/L) & W (S/T)&(H/K/Q)& (H/K/Q)& (H/K/Q)& (F/Y) (I/V/L) (F/Y/P) (W/M) N-glycosylated(Y/W/F/P) & H/P/F/W/Y HA Polypeptide MPER (R/D/H/K/E/Q/N) Sialylated(Y/W/F) H/R/Q/S Glycans (e.g., on HA receptors)

In some particular embodiments, provided infold agents have an aminoacid sequence that is substantially homologous to that of an infoldagent set forth in Table 9. In some embodiments, provided infold agentshave an amino acid sequence that is substantially identical to that ofan infold agent in Table 9.

An exemplary list of particular infold agents designed to bind HA MPER(e.g., broad spectrum, glycosylated and non-glycosylated) is provided inTable 9. According to the present invention, we find that fewer than 10%of the amino acids contribute towards HA binding. The present inventionprovides infold agents that have more than 50% pairwise sequenceidentity to any of the infold agent sequences listed below in Table 3.In particular, the present invention provides such infold agents whosestructure additionally follows rules or parameters set forth in any oneof Tables 4-8 and FIGS. 5 and 13.

TABLE 9Amino acid sequences of infolds and their binding protein/glycan targets.BINDS TO MPER- Sialylated proximal S.NO. AMINO ACID SEQUENCE HA MPERGlycans N-glycan Infold-1 MEHPVATLSTVERRAINLTWTKPFDGNSPLIRYILEM Yes YesNo SENNAPWTVLLASVDPKATSVTVKGLVPARSYQFRLCAVNDVGKGQFSKDTERVSLPE (SEQ ID NO: 1) Infold-2MPSVSDVPRDLEVVAATPTSLLISWDAPWTMSSRYY Yes No NoRITYGETGGNSPVQEFTVPGFMGGKSTATISGLKPGVDYTITVYAVYGRGDSPASSKPISINYRTEIDKPSQGGS (SEQ ID NO: 2) Infold-3MEHPVATLSTVERRAIQLTWDAPVTTSSRRYILEMSE Yes Yes NoNNAPWTVLLTVPGFMGGKTSVTVKGLVPARSYQFRLCAVNYVGKGQFSKDTERVSLPE (SEQ ID NO: 3) Infold-4MVPRDLEVVAATPTSLLISWDAPVTTSSRYYRITYGE Yes No NoTGGNSPVQEFTVPGFMGGKSTATIRGLKPGVDYTITVYAVYGRGDSPASSKPISINYRTEIDKPSQGGS (SEQ ID NO: 4) Infold-5MGSLEVVAASGADSLLISWDAPFTIYSRYYRITYHVE Yes No NoKNGSKYGPDGLPYLQEFTVPGFMGGKSTATIRNVTEDDYTITVYAVYGRGDSPASSKPISINYRTDV (SEQ ID NO: 5) Infold-6MSPSIDQVEPYSSTAQVQFKRPSRTVPIYHYKAEWRA Yes No NoVGEEVWHSKWYPFRIGGKGIVTIVGLKPETTYAVRL AAFTGSGGRSSAASEFKTQP (SEQ ID NO: 6)Infold-7 MAGSPANASTSGGDVEFTCRVFTDYPHIQWILHVEY Yes No NoLKVLTAAYKKRKETLYIRNVTEDAGEYTCLAGNNEG ISFHSAWLTVLP (SEQ ID NO: 7)Infold-8 MGSPLAPSSKSTSGGTAALGCLVKDPFTISFVTVSWN Yes No NoSGALTSGVHTPGYKKSSVVTVPSSSLGTQTYICNVNH YGKPSNTKVDKRVE (SEQ ID NO: 8)Infold-9 MVYELQVQKSVTVQEGLCVLVPCSFSSEVTFSSFYV Yes Yes NoYWFRDGGHGYYAEVVATISPMFGTPNYAPETQGRFRLLGDVQKKNCSLSIGDARMEDTGSYFFRVERGYICSGGTCRDVKYSYQQNKLNLEVTALI (SEQ ID NO: 9) Infold-10MVYELQVQKSVTVQEGLCVLVPCSFSSEVTFSSFYV Yes Yes NoYWFRDGGHGYYAEVFYTTSPGFMGGKNCSLSIGDA RMEDTGSYFFRVERGYICSGGTCRDVKYSYQQNKLNLEVT (SEQ ID NO: 10) Infold-11 MEVQLVESGGGLVKAGGSLILSCGVSNVTISSHTMN YesNo Yes WVRRVPGGGLEWVASISTMFTYRDYADAVKGRFTVSRDDLEDFVYLQMHKMRVEDTAIYYCARSPSYICSG GTCVFDAWGPGTVVTVSSGGGSGGGSGGGGIQPGMTQSPSTLSASVGDTITITCRASQSIETWLAWYQQKPGKAPKLLIYKASTLKTGVPSRFSGSGSGTEFTLTISGLQFDDFATYHCQHYAGYSATFGQGTRVEIK (SEQ ID NO: 11) Infold-12MEVQLVESGGGLVKAGGSLILSCGVSNVTISSHTMN Yes No YesWVRRVPGGGLEWVASISTMFTYRDYADAVKGRFTV SRDDLEDFVYLQMHKMRVEDTAIYYCARKGSDRLSDNDPFDAWGPGTVVTVSSGGGSGGGSGGGGIQPGMTQSPSTLSASVGDTITITCRASQSIETWLAWYQQKPGKAPKLLIYKASTLKTGVPSRFSGSGSGTEFTLTISGLQFDDFATYHCQHYAGYSATFGQGTRVEIK (SEQ ID NO: 12) Infold-13MVQLVEAGGGLVKAGGSLDLRCGVSNVTISSHTMN Yes No NoWKRRVPGGGTESVASISTMFTYTAYADAVKGRFTVS RADLEDSVSLQMHKMRVEDTAIYYCARKGSDRLSDNDPFDAWGPGTVVTVSP (SEQ ID NO: 13) Infold-14MVQLVESGGGLVGSTSSLILSCGVSNFYIHSHTMNW Yes No YesVRRAPSAGLEWVASISTFVYYRDYAQSVASAFTVSR DTRQEFVYLQMASMVAQVSAIYYCARKGSAVLSDNDPFDAWGPGTVVTVSP (SEQ ID NO: 14) Infold-15MQVQLVQSGAEVKKPGSSVKVSCTSSEVTFSSFTISW Yes No YesVRQAPGQGLEWLGGISTMFGTPNYAQKFQGRVTITA DQSTRTAYMDLRSLRSEDTAVYYCARKGSDRLSDNDPFDHWGQGTLVTVSS (SEQ ID NO: 15) Infold-16MPSVSDVPRDLEVVAATPTSLLISWATTGKASSLYYR Yes No NoITYGETGGNSPVQEFTVPAFMGGWVKATIRGLKPGVDYTITVYAVYHYGGSDDTLSPISINYRTEIDKPSQGGS (SEQ ID NO: 16) Infold-17MRDLEVVAATPTSLLISWDAPVTTSSRYYIIEMSETN Yes Yes NoAPWTVLFTVPGFMGGKSTATISGLKPGVDYTFRVCAVNYVGKGQFSKDTENVRLEI (SEQ ID NO: 17) Infold-18MRDLEVVAATPTSLLISWDAPVTTVSTYRITYGETGG Yes No YesNSPVQEFTVSTMGGTPNYAQKFQGRVTITAGTWGKSTATISGLKPGVDYTITVYRKGSDRLSDNDPSSKPISIN YRTEI (SEQ ID NO: 18) Infold-19MRDLEVVAATPTSLLISWDAPVTTVSTYYIIEMSETN Yes Yes YesAPWTVEFTVSTMGGTPNYAQKFQGRVTITAGTWG-KSTATISGLKPGVDYTFRVCAVRKGSDRLSDNDPSSK PISINYRTEI (SEQ ID NO: 19)Infold-20 MPPAVQHLTAEVTADSGEYQVLARWRYPKDRKYQS Yes No NoFLQRLTVTADDGSERLVSTARTRETTYRFTQLALGN YRLTVRAVNAWRQQGDPASVSFRIAAP (SEQ IDNO: 20) Infold-21 MGPQGFPWRLHVTGLTTSTTELAWDPPKYSEHNIFIR Yes No NoSYTVVFRDINSQQELQNITDGRGEFTLTGLKPDTTYDIKVRAWTYTRSGPLSPSIQSRTMP (SEQ ID NO: 21) Infold-22MEHPVATLSTVERRAIQLTWDAPVTTSSRRYILEMSE Yes Yes NoNNAPWTVLLTVPGFMGGKTSVTVKGLVPARSYQFRLSAVNYVGKGQYSKDTERVSLPEEPPTAPPQNVIASGRTNQSIMIQWQPPPESHQNGILKGYIIRYNNAGNPVGYQFKNITDADVNNLLLEDLTSGTNYEIEVAAYNSAG LGVYSSKVTEWTLQ (SEQ ID NO: 22)Infold-23 Chain1: EVQLVESGGGLVQPGGSLRLSCAASGFNIMD Yes No NoTYIHWVRQAPGKGLEWVARIFPLFGYTRYADSVKGR FTISARLWKNTAYLQMNSLRAEDTAVYYCSRWGGRKFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEP (SEQ ID NO: 23)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-24 Chain 1: EVQLVESGGGLVQPGGSLRLSCASSEVTFSSFAISWVYes No No RQAPGKGLEWVAGISPMFGTPNYADSVKGRFTISADQSTRTAYLQMNSLRAEDTAVYYCARSPSYICSGGTC VFDHWGQGTLVTVS (SEQ ID NO: 25)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-25 Chain 1: EVQLVESGGGLVQPGGSLRLSCASSEMTMGGSAISWYes No No VRQAPGKGLEWVAGISPMFGTPNYADSVKGRFTISADQSTRTAYLQMNSLRAEDTAVYYCARSPSYICSGGT CVFDHWGQGTLVTVS (SEQ ID NO: 26)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-26 Chain 1: Yes No NOEVQLVESGGGLVQPGGSLRLSCASSEMTMGGSAISWVRQAPGKGLEWVAGISPMFGTPNYADSVKGRFTISADGSSGTAYLQMNSLRAEDTAVYYCARSPSYICSGGT CVFDHWGQGTLVTVS (SEQ ID NO: 27)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-27 Chain 1: Yes No NoEVQLVESGGGLVQPGGSLRLSCASSEVTFSSFAISWVRQAPGKGLEWVAGISPMMGHPNYADSVKGRFTISA DQSTRTAYLQMNSLRAEDTAVYYCARSPSYICMQMTCVFDHWGQGTLVTVS (SEQ ID NO: 28)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-28 Chain 1: Yes No NoEVKLVESGGGLVQPGGSLRLSCASSEVTFSSFALTWVRQPPGKAMEWVAGISPMFGTPNYSDSVKGRFTISADQSTRTAYLQMNTLRAEDSAMYYCARSPSYICSGGTC VFDHWGQGTTVTVS (SEQ ID NO: 29)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-29 Chain 1: Yes No NoEVKLVESGGGLVQPGGSLRLSCASSEMTMGGSALT WVRQPPGKAMEWVAGISPMFGTPNYSDSVKGRFTISADQSTRTAYLQMNTLRAEDSAMYYCARSPSYICSGG TCVFDHWGQGTTVTVS (SEQ ID NO: 30)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-30 Chain 1: Yes No NoEVKLVESGGGLVQPGGSLRLSCASSEMTMGGSALT WVRQPPGKAMEWVAGISPMFGTPNYSDSVKGRFTISADGSSGTAYLQMNTLRAEDSAMYYCARSPSYICSGG TCVFDHWGQGTTVTV (SEQ ID NO: 31)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-31 Chain 1: Yes No NoEVKLVESGGGLVQPGGSLRLSCASSEVTFSSFALTWVRQPPGKAMEWVAGISPMMGHPNYSDSVKGRFTISAD QSTRTAYLQMNTLRAEDSAMYYCARSPSYICMQMTCVFDHWGQGTTVTVS (SEQ ID NO: 32) Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-32 Chain 1: Yes No NoEVKLVESGGGLVQPGGSLRLSCASSEVTFSSFALTWVRQPPGKAMEWVAGISPMFGTPNYSDSVKGRFTISADGSSGTAYLQMNTLRAEDSAMYYCARSPSYICSGGTC VFDHWGQGTTVTVS (SEQ ID NO: 33)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-33 Chain 1: Yes No NoEVKLVESGGGLVQPGGSLRLSCASSEVTFSSFALTWVRQPPGKAMEWVAGISPMFGTPNYSDSVKGRFTISAD QSTRTAYLQMNTLRAEDSAMYYCARSPSYICMQMTCVFDHWGQGTTVTVS (SEQ ID NO: 34) Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-34 Chain 1: Yes No NoEVKLVESGGGLVQPGGSLRLSCASSEVTFSSFALTWVRQPPGKAMEWVAGISPMMGHPNYSDSVKGRFTISADQSTRTAYLQMNTLRAEDSAMYYCARSPSYICSGGTC VFDHWGQGTTVTVS (SEQ ID NO: 35)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-35 Chain 1: Yes No NoEVKLVESGGGLVQPGGSLRLSCASSEMTMGGSALT WVRQPPGKAMEWVAGISPMFGTPNYSDSVKGRFTISADQSTRTAYLQMNTLRAEDSAMYYCARSPSYICSGG TCVFDHWGQGTTVTVS (SEQ ID NO: 36)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-36 Chain 1: Yes No NoEVKLVESGGGLVQPGGSLRLSCASSEMTMGGSALT WVRQPPGKAMEWVAGISPMFGTPNYSDSVKGRFTISADGSSGTAYLQMNTLRAEDSAMYYCARSPSYICSGG TCVFDHWGQGTTVTVS (SEQ ID NO: 37)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-37 Chain 1: Yes No NoEVKLVESGGGLVQPGGSLRLSCASSEVTFSSFALTWVRQPPGKAMEWVAGISPMMGHPNYSDSVKGRFTISAD QSTRTAYLQMNTLRAEDSAMYYCARSPSYICMQMTCVFDHWGQGTTVTVS (SEQ ID NO: 38) Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-38 Chain 1: Yes No NoEVKLVESGGGLVQPGGSLRLSCASSEMTMGGSALT WVRQPPGKAMEWVAGISPMMGHPNYSDSVKGRFTISADQSTRTAYLQMNTLRAEDSAMYYCARSPSYICSG GTCVFDHWGQGTTVTVS (SEQ ID NO: 39)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-39 Chain 1: Yes No NoEVKLVESGGGLVQPGGSLRLSCASSEMTMGGSALT WVRQPPGKAMEWVAGISPMMGHPNYSDSVKGRFTISADQSTRTAYLQMNTLRAEDSAMYYCARSPSYICM QMTCVFDHWGQGTTVTVS (SEQ ID NO: 40)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) Infold-40 Chain 1: Yes No NoEVKLVESGGGLVQPGGSLRLSCASSEMTMGGSALT WVRQPPGKAMEWVAGISPMMGHPNYSDSVKGRFTISADGSSGTAYLQMNTLRAEDSAMYYCARSPSYICM QMTCVFDHWGQGTTVTVS (SEQ ID NO: 41)Chain2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24)

In some embodiments, infold agents bind the MPER region of the HApolypeptide and are selected from the group comprising Infold-1,Infold-2, Infold-3, Infold-4, Infold-5, Infold-6, Infold-7, Infold-8,Infold-9, Infold-10, Infold-11, Infold-12, Infold-13, Infold-14,Infold-15, Infold-16, Infold-17, Infold-18, Infold-19, Infold-20,Infold-21, Infold-22, Infold-23, Infold-24, Infold-25, Infold-26,Infold-27, Infold-28, Infold-29, Infold-30, Infold-31, Infold-32,Infold-33, Infold-34, Infold-35, Infold-36, Infold-37, Infold-38,Infold-39, or Infold-40. In some embodiments, infold agents bind theMPER region of the HA polypeptide and are selected from the groupcomprising Infold-1, Infold-2, Infold-3, Infold-4, Infold-5, Infold-6,Infold-7, Infold-8, Infold-9, Infold-10, Infold-11, Infold-12,Infold-13, Infold-14, Infold-15, Infold-16, Infold-17, Infold-18,Infold-19, Infold-20, Infold-21, or Infold-22. In some embodiments,infold agents bind the MPER region of the HA polypeptide and areselected from the group comprising Infold-23, Infold-24, Infold-25,Infold-26, Infold-27, Infold-28, Infold-29, Infold-30, Infold-31,Infold-32, Infold-33, Infold-34, Infold-35, Infold-36, Infold-37,Infold-38, Infold-39, or Infold-40.

In some embodiments, infold agents bind the MPER region of the HApolypeptide and glycans, and are selected from the group comprisingInfold-1, Infold-3, Infold-9, Infold-10, Infold-11, Infold-12,Infold-14, Infold-15, Infold-17, Infold-18, or Infold-19. In someembodiments, infold agents bind the MPER region of the HA polypeptideand glycans, and are selected from the group comprising Infold-1,Infold-3, Infold-9, Infold-10, Infold-11, Infold-12, Infold-14,Infold-15, Infold-17, Infold-18, Infold-19, or Infold-22.

In some embodiments, infold agents bind the MPER region of the HApolypeptide and the MPER-proximal N-glycans on the HA polypeptide, andare selected from the group comprising Infold-11, Infold-12, Infold-14,Infold-15, Infold-18 or Infold-19.

In some embodiments, infold agents bind the MPER region of the HApolypeptide and sialylated glycans on the HA receptor, and are selectedfrom the group comprising Infold-1, Infold-3, Infold-9, Infold-10,Infold-17, or Infold-19. In some embodiments, infold agents bind theMPER region of the HA polypeptide and sialylated glycans on the HAreceptor, and are selected from the group comprising Infold-1, Infold-3,Infold-9, Infold-10, Infold-17, Infold-19, or Infold-22.

In further embodiments, infold agents bind the MPER region of the HApolypeptide, the MPER-proximal N-glycans on the HA polypeptide andsialylated glycans on the HA receptor and is Infold-19.

In some embodiments, infold agents for use in accordance with thepresent invention include any of those presented in Table 9. In someembodiments, infold agents are selected from the group comprisingInfold-1, Infold-3, Infold-9, Infold-10, Infold-11, Infold-12,Infold-14, Infold-15, Infold-17, Infold-18, Infold-19, Infold 22,Infold-28, and Infold-34. In some embodiments, infold agents areselected from the group comprising Infold-1, Infold-3, Infold-9,Infold-10, Infold-17, Infold-19 and Infold-28.

In some embodiments, the cognate target of infold agents in accordancewith the present disclosure include at least one that corresponds to aresidue found in HA at a position selected from the group consisting of:18, 19, 20, 21, 41, 45, 49, 52, 53 and 56, and combinations thereof. Insome embodiments, the cognate target residues include at least one thatcorresponds to a residue selected from the group consisting of Trp21,Ile45, Asp19, Asn19, Ala19, His18, Gln18, Leu18, Ile18, Va118, Gly20,Thr41, Thr49, Asn49, Gln49, Va152, Leu52, Ile52, Asn53, Ile56 and Va156.

In some embodiments, when the cognate target residues include at leastone that corresponds to a residue found in HA at position 18, the infoldagent is arranged and constructed such that it contains an interactionresidue selected from the group consisting of His, Asp, Glu, Trp, Tyr,Asn, Lys, Arg, Gln, Met, Cys, Phe, Ile, Leu, and Val that is positionedwith a 4-7 A radius of the target residue corresponding to HA position18 when the infold agent is in the vicinity of the binding site. In someembodiments, when the cognate target residues include at least one thatcorresponds to a residue found in HA at position 18, the infold agent isarranged and constructed such that it contains an interaction residueselected from the group consisting of His, Asp, Glu, Trp, Tyr, Asn, Lys,Arg, Gln, Met, Cys, Phe, Ile, Leu, Val, Thr, Ser, Gly, Ala, and Pro thatis positioned with a 4-7 A radius of the target residue corresponding toHA position 18 when the infold agent is in the vicinity of the bindingsite. In some embodiments, when the cognate target residues include atleast one that corresponds to a residue found in HA at position 18, theinfold agent does not contain a residue other than His, Asp, Glu, Trp,Tyr, Asn, Lys, Arg, Gln, Met, Cys, Phe, Ile, Leu, Val, Thr, Ser, Gly,Ala, or Pro when that is positioned within a 4-7 A radius when theinfold agent is in the vicinity of the binding site.

In some embodiments, when the cognate target residues include at leastone that corresponds to a residue found in HA at position 19, the infoldagent is arranged and constructed such that it contains an interactionresidue selected from the group consisting of Arg, Lys, His, Ser, Thr,Asn, Asp, Gln, Glu, Ile, Val, Ala, and Gly that is positioned with a 4-7A radius of the target residue corresponding to HA position 19 when theinfold agent is in the vicinity of the binding site. In someembodiments, when the cognate target residues include at least one thatcorresponds to a residue found in HA at position 19, the infold agent isarranged and constructed such that it contains an interaction residueselected from the group consisting of Arg, Lys, His, Ser, Thr, Asn, Asp,Gln, Glu, Ile, Val, Ala, Gly, Tyr, Pro, Trp, Phe, Leu, Cys, and Met thatis positioned with a 4-7 A radius of the target residue corresponding toHA position 19 when the infold agent is in the vicinity of the bindingsite. In some embodiments, when the cognate target residues include atleast one that corresponds to a residue found in HA at position 19, theinfold agent does not contain a residue other than Arg, Lys, His, Ser,Thr, Asn, Asp, Gln, Glu, Ile, Val, Ala, Gly, Tyr, Pro, Trp, Phe, Leu,Cys, or Met when that is positioned within a 4-7 A radius when theinfold agent is in the vicinity of the binding site.

In some embodiments, when the cognate target residues include at leastone that corresponds to a residue found in HA at position 20, the infoldagent is arranged and constructed such that it contains an interactionresidue selected from the group consisting of Gly, Ala, Cys, Met, Serand Pro that is positioned with a 4-7 A radius of the target residuecorresponding to HA position 20 when the infold agent is in the vicinityof the binding site. In some embodiments, when the cognate targetresidues include at least one that corresponds to a residue found in HAat position 20, the infold agent is arranged and constructed such thatit contains an interaction residue selected from the group consisting ofGly, Ala, Asn, Asp, Arg, Phe, Trp, His, Tyr, Gln, and Lys that ispositioned with a 4-7 A radius of the target residue corresponding to HAposition 20 when the infold agent is in the vicinity of the bindingsite. In some embodiments, when the cognate target residues include atleast one that corresponds to a residue found in HA at position 20, theinfold agent does not contain a residue other than Gly, Ala, Cys, Met,Ser, Pro, Asn, Asp, Arg, Phe, Trp, His, Tyr, Gln, or Lys when that ispositioned within a 4-7 A radius when the infold agent is in thevicinity of the binding site.

In some embodiments, when the cognate target residues include at leastone that corresponds to a residue found in HA at position 21, the infoldagent is arranged and constructed such that it contains an interactionresidue selected from the group consisting of Tyr, Ile, Met, Phe, His,Cys, and Pro that is positioned with a 4-7 A radius of the targetresidue corresponding to HA position 21 when the infold agent is in thevicinity of the binding site. In some embodiments, when the cognatetarget residues include at least one that corresponds to a residue foundin HA at position 21, the infold agent is arranged and constructed suchthat it contains an interaction residue selected from the groupconsisting of Gly, Val, Arg, Ser, Thr, Trp, Leu, and Ala that ispositioned with a 4-7 A radius of the target residue corresponding to HAposition 21 when the infold agent is in the vicinity of the bindingsite. In some embodiments, when the cognate target residues include atleast one that corresponds to a residue found in HA at position 21, theinfold agent does not contain a residue other than Tyr, Ile, Met, Phe,His, Cys, Pro, Gly, Val, Arg, Ser, Thr, Trp, Leu, or Ala when that ispositioned within a 4-7 A radius when the infold agent is in thevicinity of the binding site.

In some embodiments, when the cognate target residues include at leastone that corresponds to a residue found in HA at position 41, the infoldagent is arranged and constructed such that it contains an interactionresidue selected from the group consisting of Ser, Thr, Asp, Asn. Glu,and Gln that is positioned with a 4-7 A radius of the target residuecorresponding to HA position 41 when the infold agent is in the vicinityof the binding site. In some embodiments, when the cognate targetresidues include at least one that corresponds to a residue found in HAat position 41, the infold agent is arranged and constructed such thatit contains an interaction residue selected from the group consisting ofMet, Ile, Val, Tyr, Ala, Gly, His, Arg, Lys, and Pro that is positionedwith a 4-7 A radius of the target residue corresponding to HA position41 when the infold agent is in the vicinity of the binding site. In someembodiments, when the cognate target residues include at least one thatcorresponds to a residue found in HA at position 41, the infold agentdoes not contain a residue other than Ser, Thr, Asp, Asn. Glu, Gln, Met,Ile, Val, Tyr, Ala, Gly, His, Arg, Lys, or Pro when that is positionedwithin a 4-7 A radius when the infold agent is in the vicinity of thebinding site.

In some embodiments, when the cognate target residues include at leastone that corresponds to a residue found in HA at position 45, the infoldagent is arranged and constructed such that it contains an interactionresidue selected from the group consisting of Ile, Met, Phe, Leu, Val,Trp, and Cys that is positioned with a 4-7 A radius of the targetresidue corresponding to HA position 45 when the infold agent is in thevicinity of the binding site. In some embodiments, when the cognatetarget residues include at least one that corresponds to a residue foundin HA at position 45, the infold agent is arranged and constructed suchthat it contains an interaction residue selected from the groupconsisting of Tyr, Pro, Ala, and Thr that is positioned with a 4-7 Aradius of the target residue corresponding to HA position 45 when theinfold agent is in the vicinity of the binding site. In someembodiments, when the cognate target residues include at least one thatcorresponds to a residue found in HA at position 45, the infold agentdoes not contain a residue other than Ile, Met, Phe, Leu, Val, Trp, Cys,Tyr, Pro, Ala or Thr when that is positioned within a 4-7 A radius whenthe infold agent is in the vicinity of the binding site.

In some embodiments, when the cognate target residues include at leastone that corresponds to a residue found in HA at position 49, the infoldagent is arranged and constructed such that it contains an interactionresidue selected from the group consisting of Ser, Thr, Asp, Asn, Glu,Gln, Lys, and Arg that is positioned with a 4-7 A radius of the targetresidue corresponding to HA position 45 when the infold agent is in thevicinity of the binding site. In some embodiments, when the cognatetarget residues include at least one that corresponds to a residue foundin HA at position 49, the infold agent is arranged and constructed suchthat it contains an interaction residue selected from the groupconsisting of Met, Ile, Val, Tyr, Ala, Gly, His, Arg, Lys, Pro, Trp, Serand Thr that is positioned with a 4-7 A radius of the target residuecorresponding to HA position 49 when the infold agent is in the vicinityof the binding site. In some embodiments, when the cognate targetresidues include at least one that corresponds to a residue found in HAat position 49, the infold agent does not contain a residue other thanSer, Thr, Asp, Asn, Glu, Gln, Lys, Arg, Met, Ile, Val, Tyr, Ala, Gly,His, Pro, or Trp when that is positioned within a 4-7 A radius when theinfold agent is in the vicinity of the binding site.

In some embodiments, when the cognate target residues include at leastone that corresponds to a residue found in HA at position 52, the infoldagent is arranged and constructed such that it contains an interactionresidue selected from the group consisting of Val, Leu, Ile, Phe, Met,Cys, Tyr, and Trp that is positioned with a 4-7 A radius of the targetresidue corresponding to HA position 52 when the infold agent is in thevicinity of the binding site. In some embodiments, when the cognatetarget residues include at least one that corresponds to a residue foundin HA at position 52, the infold agent is arranged and constructed suchthat it contains an interaction residue selected from the groupconsisting of Cys, Met, Trp, Tyr, Ala, Gly, Thr, Pro, His, Ser, and Aspthat is positioned with a 4-7 A radius of the target residuecorresponding to HA position 52 when the infold agent is in the vicinityof the binding site. In some embodiments, when the cognate targetresidues include at least one that corresponds to a residue found in HAat position 52, the infold agent does not contain a residue other thanVal, Leu, Ile, Phe, Met, Cys, Tyr, Trp, Ala, Gly, The, Pro, His or Serwhen that is positioned within a 4-7 A radius when the infold agent isin the vicinity of the binding site.

In some embodiments, when the cognate target residues include at leastone that corresponds to a residue found in HA at position 53, the infoldagent is arranged and constructed such that it contains an interactionresidue selected from the group consisting of Asn, Asp, Gln, Glu, Ser,Thr, and Lys that is positioned with a 4-7 A radius of the targetresidue corresponding to HA position 53 when the infold agent is in thevicinity of the binding site. In some embodiments, when the cognatetarget residues include at least one that corresponds to a residue foundin HA at position 53, the infold agent is arranged and constructed suchthat it contains an interaction residue selected from the groupconsisting of His, Arg, Tyr, Gly, Ala, Trp, and Pro that is positionedwith a 4-7 A radius of the target residue corresponding to HA position53 when the infold agent is in the vicinity of the binding site. In someembodiments, when the cognate target residues include at least one thatcorresponds to a residue found in HA at position 53, the infold agentdoes not contain a residue other than Asn, Asp, Gln, Glu, Ser, Thr, Lys,His, Arg, Tyr, Gly, Ala, Trp or Pro when that is positioned within a 4-7A radius when the infold agent is in the vicinity of the binding site.

In some embodiments, when the cognate target residues include at leastone that corresponds to a residue found in HA at position 56, the infoldagent is arranged and constructed such that it contains an interactionresidue selected from the group consisting of Ile, Met, Phe, Leu, Val,Trp, and Cys that is positioned with a 4-7 A radius of the targetresidue corresponding to HA position 56 when the infold agent is in thevicinity of the binding site. In some embodiments, when the cognatetarget residues include at least one that corresponds to a residue foundin HA at position 56, the infold agent is arranged and constructed suchthat it contains an interaction residue selected from the groupconsisting of Tyr, Pro, Ala, Thr, Cys, Met, Trp, and Gly that ispositioned with a 4-7 A radius of the target residue corresponding to HAposition 56 when the infold agent is in the vicinity of the bindingsite. In some embodiments, when the cognate target residues include atleast one that corresponds to a residue found in HA at position 56, theinfold agent does not contain a residue other than Ile, Met, Phe, Leu,Val, Trp, Cys, Tyr, Pro, Ala, Thr, Trp, or Gly when that is positionedwithin a 4-7 A radius when the infold agent is in the vicinity of thebinding site.

As discussed further below, in some embodiments, an infold agent is apolypeptide that binds to a selected binding site. In many embodiments,an infold agent has a structure characterized by a “fold” backbonepopulated by interaction residues selected and arranged so that, whenthe infold agent is in the vicinity of the binding site, individualinteraction residues are positioned within a preselected distance orvolume of cognate target residues. In some embodiments, an infold agentis an engineered or designed polypeptide. In some embodiments, infoldagents provided herein bind a hemagglutinin (HA) polypeptide. In someembodiments, infold agents bind to an HA polypeptide in its MPER region.In some embodiments, infold agents bind to an HA polypeptide MPER regionindependent of its glycosylation. For example, in some embodiments,infold agents are designed to be of appropriate size that their bindingto an MPER region is not prevented by its glycosylation. In someembodiments, an infold agent binds to a glycosylated MPER region with anaffinity that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of its affinity foran otherwise identical non-glycosylated MPER region. In someembodiments, infold agents have volumetric sizes between 6000-1,20,000Å³. In some embodiments, provided infold agents have a volumetric sizethat is equal to or less than the volumetric size of an antibody. Insome embodiments, an infold agent has a total target epitope surfacearea of approximately 20×30=600 Å². In some embodiments, the totaltarget epitope surface area of an infold agent is less than about 10 Å²,20 Å², 30 Å², 40 Å², 50 Å², 60 Å², 70 Å², 80 Å², 85 Å², 90 Å², 95 Å²,100 Å², 105 Å², 110 Å², 115 Å², 120 Å², 125 Å², 130 Å², 135 Å², 140 Å²,145 Å², 150 Å², 151 Å², 152 Å², 153 Å², 154 Å², 155 Å², 160 Å², 165 Å²,170 Å², 175 Å², 180 Å², 185 Å², 190 Å², 195 Å², 200 Å², 210 Å², 220 Å²,230 Å², 240 Å², 250 Å², 260 Å², 270 Å², 280 Å², 290 Å², 300 Å², 310 Å²,315 Å², 320 Å², 325 Å², 330 Å² or larger. In some embodiments, totaltarget epitope surface area is less than about 200 Å², about 175 Å²,about 150 Å², about 125 Å² or smaller.

In many embodiments, infold agents have a length that is less than about1000 amino acids. In some embodiments, infold agents have a length thatis less than a maximum length of about 1000, 975, 950, 925, 900, 875,850, 825, 800, 775, 750, 725, 700, 675, 650, 625, 600, 575, 550, 525,500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 240, 230, 220,210, 200, 190, 180, 170, 160, 150, 140, 130, 125, 120, 115, 110, 105,100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, or 20amino acids in length. In some embodiments, infold agents have a lengththat is greater than a minimum length of about 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or more aminoacids in length. In some embodiments, infold agents have a lengthbetween any one of such minimum lengths and any one of such maximumlengths, so long as the maximum length is longer than the minimumlength. In some particular embodiments, an infold agent has a lengthbetween about 20 and 500, or between 30 and 400, or between 40 and 300,or between 80 and 250 amino acids. In some embodiments, an infold agenthas a length of about 84, 88, 93, 95, 98, 104, 106, 110, 111, 116, 119,123, 124, 132, 212, 215, 244, or 245. In some embodiments, infold agentsare comprised of natural amino acids. In other embodiments, infoldagents are comprised of unnatural amino acids. In some embodiments,infold agents are comprised of combinations of natural and unnaturalamino acids. In some embodiments, an infold agent is comprised of one,two or more polypeptide chains that are covalently (e.g., by means of alinker) or non-covalently associated. In some embodiments, an infoldagent may be linked to, or part of, a longer polypeptide chain (e.g., acomplete antibody, serum albumin, or other carrier protein) so long asthe infold agent retains its three-dimensional structure and arrangementfor interaction. In some embodiments, infold agents may be appended tothe N- or C-termini of another polypeptide sequence that is or is not aninfold. In some embodiments, infold agents are incorporated into thesequence of another polypeptide that is or is not an infold, therebyseparating the polypeptide sequence into two or more segments. In someembodiments, appending the infold to the N or C termini or within thesequence of another polypeptide that is or is not an infold may allowfor at least one or more of the following: a decrease in immunogenicity,increased circulation lifetime, slower in vivo degradation, incitinglocal immune response, interaction with the immune system molecules, anincrease in volume, an increase in affinity for the infold target(s), anincrease in specificity for the infold target(s), or the use of othercommonly used therapeutic/prophylactic delivery protocols. In someembodiments, appending an infold to the N or C termini or within thesequence of another polypeptide that is or is not an infold does nothave a direct effect on binding of an infold agent to a target (e.g., anHA polypeptide, the MPER region of an HA polypeptide, N-glycans on an HApolypeptide, HA receptors or sialylated glycans on HA receptors).

In some embodiments, infold agents bind to their target binding sites byinteraction with one or more target residues. In some embodiments, suchtarget residues are amino acids, saccharides, or combinations thereof.In some embodiments the present invention provides, infold agents thatbind to an HA polypeptide, N-linked glycans on an HA polypeptide, an HAreceptor, sialylated glycans on an HA receptor or various combinationsthereof. In some embodiments, the present invention provides polypeptideagents comprising a first infold agent that binds to an HA polypeptideand a second infold agent that binds to the HA receptor. In some suchembodiments, the polypeptide agent comprises a single polypeptide chainthat comprises the first and second infold, optionally connected to oneanother by way of one or more linking amino acids. In some embodiments,an infold agent that binds to an HA receptor interacts with one or moreglycans on the HA receptor. In some embodiments, infold agents bindsialylated glycans. In some embodiments, infold agents bind sialylatedglycans having an umbrella-like topology. In certain embodiments, infoldagents bind to umbrella-topology glycans with high affinity and/orspecificity. In some embodiments, infold agents show a bindingpreference for umbrella-topology glycans as compared with glycans ofother topologies (e.g., cone-topology glycans). In some embodiments,infold agents compete with HA for binding to HA receptors. In someembodiments, infold agents compete with HA for binding to glycans on HAreceptors. In some embodiments, infold agents compete with HA forbinding to umbrella-topology glycans. In some embodiments, an infoldagent provided herein is an umbrella topology blocking agent. In someembodiments, an infold agent provided herein is an umbrella topologyspecific blocking agent. In some embodiments, an infold agent has abackbone fold structure populated by a plurality of direct binding aminoacid residues (i.e., amino acid residues that make direct contacts withHA amino acids or glycans), and/or with HA receptor amino acids orglycan as described herein.

Infold Agent Activities

As discussed herein, the present invention provides infold agents thatbind to HA polypeptides and/or to HA receptors. In some embodiments,provided infold agents bind to HA polypeptides independent of subtype.In some embodiments, provided infold agents that achieve universalinfluenza neutralization via binding to the HA polypeptide.

In some embodiments, infold agents bind to HA polypeptides of subtypeH1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and/orH16. Specifically, in some embodiments, infold agents bind to HApolypeptides that have sequence elements characteristic of one or moreof H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 andH16 HA polypeptides. In some embodiments, an infold agent binds to oneor more of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15 and H16 HA polypeptides with an affinity that is at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90% or more of its affinity for one or more of a different H1, H2,H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16 HApolypeptides. In some embodiments an infold agent shows bindingaffinities for different HA polypeptides (e.g., HA polypeptides fromdifferent groups, clades, or clusters and/or from different strains)that are within 5 fold binding affinity of one another. In someembodiments an infold agent shows binding affinities for different HApolypeptides that are within 2 fold of one another (see for example,FIG. 7). In some embodiments an infold agent shows binding affinitiesfor different HA polypeptides (e.g., HA polypeptides from differentgroups, clades, or clusters and/or from different strains) that arewithin 150 fold (e.g., within 100 fold, within 50 fold, within 25 fold,within 10 fold, or within 5 fold) binding affinity of one another.

In some embodiments, provided infold agents bind to at least two of H1,H3, H5, H7, and/or H9 HA polypeptides. In some embodiments, providedinfold agents bind to at least three, four or five of the H1, H3, H5,H7, and/or H9 HA polypeptides.

In some embodiments, provided infold agents bind to HA polypeptides ofat least one of subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11,H12, H13, H14, H15, and/or H16, and do not bind to at least one HApolypeptide of subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11,H12, H13, H14, H15, and/or H16. In some embodiments, infold agents bindto HA polypeptides of subtype H1. In some embodiments, infold agentsbind to HA polypeptides of subtype H1 with an affinity at least 100%, atleast 125%, at least 150%, at least 200% or more of that with which itbinds to HA polypeptides of at least one subtype H2, H3, H4, H5, H6, H7,H8, H9, H10, H11, H12, H13, H14, H15, and/or H16. In some embodiments,infold agents bind to HA polypeptides of subtype H3. In someembodiments, infold agents bind to HA polypeptides of subtype H3 with anaffinity at least 100%, at least 125%, at least 150%, at least 200% ormore of that with which it binds to HA polypeptides of at least onesubtype H1, H2, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15,and/or H16.

In some embodiments, infold agents bind to a binding site that includesregions of the HA-1 and HA-2 domains in an HA polypeptide. In someembodiments, infold agents bind regions of an HA-1 domain. In someembodiments, infold agents bind regions of HA-2 domain. In someembodiments, infold agents bind both regions of the HA-1 domain and theHA-2 domain. In some embodiments, infold agents bind the MPER region ofan HA polypeptide.

In some embodiments, infold agents bind a glycosylated MPER region. Insome embodiments, infold agents bind a non-glycosylated MPER regions. Insome embodiments, infold agents bind the MPER region of the HApolypeptide, independent of MPER glycosylation levels. In someembodiments, infold agents bind HA polypeptides independent ofglycosylation levels with high affinity and/or specificity. In someembodiments, an infold agent binds to a glycosylated MPER region with anaffinity that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of its affinity foran otherwise identical non-glycosylated MPER region.

In some embodiments, infold agents bind the HA polypeptide. In someembodiments, infold agents bind N-linked glycans on the HA polypeptide.In some embodiments, infold agents bind the MPER-proximal N-glycans onthe HA polypeptide. In some embodiments, infold agents bind N-linkedglycans in the MPER proximal region of HA polypeptides with highaffinity and/or specificity.

In some embodiments, infold agents bind HA receptors. In someembodiments, infold agents bind to both HA polypeptides and HAreceptors. In some such embodiments, one or more provided infold agentscan bind simultaneously to HA polypeptides and HA receptors. Among otherthings, the present invention encompasses the recognition that use ofinfold agents that bind to both HA polypeptides and HA receptors maypermit effective inhibition of influenza infection with lower amounts oftherapeutic agent (i.e., infold agent) than would be required for anagent that binds to only HA polypeptide or HA receptor but not both.Without wishing to be bound by any particular theory, we propose thatability to bind both sides of the HA polypeptide-HA receptor interactionpermits increased local concentration of inhibitor (i.e., infold agent)only in those sites that are relevant; infold agent is not “wasted” onHA polypeptides or receptors that are not participating in infection.

In some embodiments, infold agents bind sialylated HA receptors. In someembodiments, infold agents bind to α2,6 sialylated glycans; in someembodiments, infold agents bind preferentially to α2,6 sialylatedglycans. In certain embodiments, infold agents bind to a plurality ofdifferent α2,6 sialylated glycans. In some embodiments, infold agentsare not able to bind to α2,3 sialylated glycans, and In some embodimentsinfold agents are able to bind to α2,3 sialylated glycans.

In some embodiments, infold agents bind to sialylated glycans having anumbrella-like topology. In some embodiments, infold agents bind toumbrella topology glycans (and/or to umbrella topology glycan mimics)more strongly than they bind to cone topology glycans. In someembodiments, infold agents show a relative affinity for umbrella glycansverses cone glycans that is about 10, 9, 8, 7, 6, 5, 4, 3, or 2.

In some embodiments, infold agents bind to umbrella topology glycans(e.g., long α2,6 sialylated glycans such as, for example,Neu5Acα2,6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc) with high affinity. Forexample, in some embodiments, infold agents bind to umbrella topologyglycans with an affinity comparable to that observed for a wild type HApolypeptide that mediates infection of a humans (e.g., H1N1 HApolypeptide or H3N2 HA polypeptide). In some embodiments, infold agentsbind to umbrella glycans with an affinity that is at least 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% of that observed under comparable conditions fora wild type HA polypeptide that mediates infection of humans. In someembodiments, infold agents bind to umbrella glycans with an affinitythat is greater than that observed under comparable conditions for awild type HA polypeptide that mediates infection of humans.

In some embodiments, infold agents bind to one or more of the glycansillustrated in FIG. 12. In some embodiments, infold agents bind tomultiple glycans illustrated in FIG. 12. In some embodiments, infoldagents bind with high affinity and/or specificity to glycans illustratedin FIG. 12. In some embodiments, infold agents bind to glycansillustrated in FIG. 12 preferentially as compared with their binding toglycans illustrated in FIG. 11. In some embodiments, infold agents bindto an oligosaccharide of the following form:

Neu5Acα2,6Sug1-Sug2-Sug3

where:

-   -   1. Neu5Ac α2,6 is always or almost always at the non-reducing        end;    -   2. Sug1:        -   a. is a hexose (frequently Gal or Glc) or hexosamine (GlcNAc            or GalNAc) in α or β configuration (frequently β- for N- and            O-linked extension and α- in the case of GalNAcα- that is            O-linked to glycoprotein);        -   b. no sugars other than Neu5Acα2,6 should be attached to any            of the non-reducing positions of Sug1 (except when Sug1 is            GalNAcα- that is O-linked to the glycoprotein); and/or        -   c. non-sugar moieties such as sulfate, phosphate, guanidium,            amine, N-acetyl, etc. can be attached to non-reducing            positions (typically 6 position) of Sug1 to improve contacts            with HA;    -   3. Sug2 and/or Sug3:        -   a. hexose (frequently Gal or Glc) or hexosamine (GlcNAc or            GalNAc) in α or β configuration (frequently β); and/or        -   b. sugars (such as Fuc) or non-sugar moieties such as            sulfate, phosphate, guanidium, amine, N-acetyl, etc. can be            attached to non-reducing positions of Sug2, Sug3, and/or            Sug4;    -   4. Linkage between any two sugars in the oligosaccharide apart        from Neu5Acα2,6 linkage can be 1-2, 1-3, 1-4, and/or 1-6        (typically 1-3 or 1-4); and/or    -   5. Structure where Neu5Acα2,6 is linked GalNAcα that is O-linked        to the glycoprotein and additional sugars are linked to the        non-reducing end of GalNAcα for example        -   i. Neu5Acα2,6(Neu5Acα2,3Galβ1-3)GalNAcα-        -   ii. Neu5Acα2,6(Galβ1-3)GalNAcα-

In certain embodiments, infold agents bind to umbrella-topology glycanswith high affinity and/or specificity. The present invention encompassesthe recognition that gaining an ability to bind umbrella topologyglycans (e.g., long α2,6 sialylated glycans), and particularly anability to bind with high affinity, may confer upon an infold agent theability to provide targeted broad spectrum neutralization againstinfluenza virus. Without wishing to be bound by any particular theory,we propose that binding to umbrella topology glycans may be paramount,and in particular that loss of binding to other glycan types may not berequired.

In some embodiments, the present invention provides infold agents thatbind to umbrella topology glycans found on HA receptors of a particulartarget species. For example, in some embodiments, the present inventionprovides infold agents that bind to umbrella topology glycans found onhuman HA receptors, e.g., HA receptors found on human epithelial cells,and particularly infold agents that bind to umbrella topology glycansfound on human HA receptors in the upper respiratory tract.

In some embodiments, infold agents bind to receptors found on humanupper respiratory epithelial cells. In certain embodiments, infoldagents bind to HA receptors in the bronchus and/or trachea. In someembodiments, infold agents are not able to bind receptors in the deeplung, and in some embodiments, infold agents are able to bind receptorsin the deep lung.

In some embodiments, infold agents bind to at least about 10%, 15%, 20%,25%, 30% 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%or more of the glycans found on HA receptors in human upper respiratorytract tissues (e.g., epithelial cells).

In certain embodiments, binding affinity of infold agents is assessedover a range of concentrations. Such a strategy provides significantlymore information, particularly in multivalent binding assays, than dosingle-concentration analyses. In some embodiments, for example, bindingaffinities of infold agents are assessed over concentrations rangingover at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fold.

Production of Infold Agents

Infold agents, and/or characteristic portions thereof, or nucleic acidsencoding them, may be produced by any available means.

Infold agents (or characteristic portions) may be produced, for example,by utilizing a host cell system engineered to express an inventivepolypeptide-encoding nucleic acid. Alternatively or additionally, infoldagents may be partially or fully prepared by chemical synthesis.

Where infold agents are expressed in cells (e.g., engineered cells), anyexpression system can be used to produce infold agents (orcharacteristic portions thereof). To give but a few examples, knownexpression systems include, for example, egg, baculovirus, plant, yeast,Madin-Darby Canine Kidney cells (MDCK), or Vero (African green monkeykidney) cells. Alternatively or additionally, infold agents (orcharacteristic portions) can be expressed in cells using recombinanttechniques, such as through the use of an expression vector (Sambrook etal., Molecular Cloning: A Laboratory Manual, CSHL Press, 1989).

Alternatively or additionally, infold agents (or characteristic portionsthereof), may be produced in the context of intact virus, whetherotherwise wild type, attenuated, killed, etc. Infold agents, orcharacteristic portions thereof, may also be produced in the context ofvirus like particles.

Also, it will be appreciated by those of ordinary skill in the art thatpolypeptides, and particularly infold agents as described herein, may begenerated, identified, isolated, and/or produced by culturing cells ororganisms that produce infold agents (whether alone or as part of acomplex, including as part of a virus particle or virus), underconditions that allow ready screening and/or selection of polypeptidescapable of binding to umbrella-topology glycans. To give but oneexample, in some embodiments, it may be useful to produce and/or study acollection of infold agents under conditions that reveal and/or favorthose variants that bind to HA polypeptides or umbrella topology glycans(e.g., with particular specificity and/or affinity). In someembodiments, such a collection of infold agents results from evolutionin nature. In some embodiments, such a collection of infold agentsresults from engineering. In some embodiments, such a collection ofinfold agents results from a combination of engineering and naturalevolution.

Nucleic Acids

In certain embodiments, the present invention provides nucleic acidswhich encode an infold agent or a characteristic or biologically activeportion of an infold agent. In some embodiments, the invention providesnucleic acids which are complementary to nucleic acids which encode aninfold agent or a characteristic or biologically active portion of aninfold agent.

In some embodiments, the invention provides nucleic acid molecules whichhybridize to nucleic acids encoding an infold agent or a characteristicor biologically active portion of an infold agent. Such nucleic acidscan be used, for example, as primers or as probes. To give but a fewexamples, such nucleic acids can be used as primers in polymerase chainreaction (PCR), as probes for hybridization (including in situhybridization), and/or as primers for reverse transcription-PCR(RT-PCR).

In certain embodiments, nucleic acids can be DNA or RNA, and can besingle stranded or double-stranded. In some embodiments, nucleic acidsmay include one or more non-natural nucleotides; In some embodiments,nucleic acids include only natural nucleotides.

Antibodies to Infold Agents

The present invention provides antibodies to infold agents. These may bemonoclonal or polyclonal and may be prepared by any of a variety oftechniques known to those of ordinary skill in the art (e.g., see Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988). For example, antibodies can be produced by cellculture techniques, including the generation of monoclonal antibodies,or via transfection of antibody genes into suitable bacterial ormammalian cell hosts, in order to allow for the production ofrecombinant antibodies.

Systems for Identifying and/or Characterizing Infold Agents

The present invention provides a variety of systems for testing,characterizing, and/or identifying infold agents.

In some embodiments, infold agents are characterized by such systems andmethods that involve contacting the infold agent with one or morecandidate substrates, such as regions of HA polypeptides, the MPERregion of HA-polypeptides, N-glycans on HA polypeptides, HA receptors,sialylated HA receptors, glycans on sialylated HA receptors and/orumbrella topology glycans on sialylated HA receptors.

In some embodiments, an infold agent and/or candidate substrate may befree in solution, fixed to a support, and/or expressed in and/or on thesurface of a cell. The candidate substrate and/or infold agent may belabeled, thereby permitting detection of binding. Either the infoldagent or the candidate substrate is the labeled species. Competitivebinding formats may be performed in which one of the substances islabeled, and one may measure the amount of free label versus bound labelto determine the effect on binding.

In some embodiments, binding assays involve, for example, exposing acandidate substrate to an infold agent and detecting binding between thecandidate substrate and the infold agent. A binding assay may beconducted in vitro (e.g., in a candidate tube, comprising substantiallyonly the components mentioned; in cell-free extracts; and/or insubstantially purified components). Alternatively or additionally,binding assays may be conducted in cyto and/or in vivo (e.g., within acell, tissue, organ, and/or organism; described in further detailbelow).

In certain embodiments, at least one infold agent is contacted with atleast one candidate substrate and an effect detected. In someembodiments, for example, an infold agent is contacted with a candidatesubstrate, and binding between the two entities is monitored. In someembodiments, an assay may involve contacting a candidate substrate witha characteristic portion of an infold agent. Binding of the infold agentto the candidate substrate is detected. It will be appreciated thatfragments, portions, homologs, variants, and/or derivatives of infoldagents may be employed, provided that they comprise the ability to bindone or more candidate substrates.

Binding of a infold agent to the candidate substrate may be determinedby a variety of methods well-known in the art. The present inventionprovides assays involving solid phase-bound infold agents and detectingtheir interactions with one or more candidate substrates. Thus, aninfold agent may comprise a detectable marker, such as a radioactive,fluorescent, and/or luminescent label. Furthermore, candidate substratecan be coupled to substances which permit indirect detection (e.g. bymeans of employing an enzyme which uses a chromogenic substrate and/orby means of binding a detectable antibody). Changes in the conformationof infold agents as the result of an interaction with a candidatesubstrate may be detected, for example, by the change in the emission ofthe detectable marker. Alternatively or additionally, solid phase-boundprotein complexes may be analyzed by means of mass spectrometry.

In some embodiments, the infold agent can be non-immobilized. In someembodiments, the non-immobilized component may be labeled (with forexample, a radioactive label, an epitope tag, an enzyme-antibodyconjugate, etc.). Alternatively or additionally, binding may bedetermined by immunological detection techniques. For example, thereaction mixture may be subjected to Western blotting and the blotprobed with an antibody that detects the non-immobilized component.Alternatively or additionally, enzyme linked immunosorbent assay (ELISA)may be utilized to assay for binding.

In certain embodiments, cells may be directly assayed for bindingbetween infold agents and candidate substrates. Immunohistochemicaltechniques, confocal techniques, and/or other techniques to assessbinding are well known to those of skill in the art. Various cell linesmay be utilized for such screening assays, including cells specificallyengineered for this purpose. Examples of cells used in the screeningassays include mammalian cells, fungal cells, bacterial cells, or viralcells. A cell may be a stimulated cell, such as a cell stimulated with agrowth factor. One of skill in the art would understand that theinvention disclosed herein contemplates a wide variety of in cyto assaysfor measuring the ability of infold agents to bind to candidatesubstrates.

Depending on the assay, cell and/or tissue culture may be required. Acell may be examined using any of a number of different physiologicassays. Alternatively or additionally, molecular analysis may beperformed, including, but not limited to, western blotting to monitorprotein expression and/or test for protein-protein interactions; massspectrometry to monitor other chemical modifications; etc.

The present invention provides methods for identifying infold agentsthat bind to candidate substrates and, therefore, may be involved ininfluenza infection. One in cyto method of identifying substances thatbind to candidate substrates is the two-hybrid system assay (Fields etal., 1994, Trends in Genetics, 10:286; and Colas et al., 1998, TIBTECH,16:355; both of which are incorporated herein by reference). In thisassay, yeast cells express a first fusion protein comprising a testsubstance in accordance with the present invention (e.g. an infoldagent, gene encoding an infold agent, and/or characteristic portionsthereof) and a DNA-binding domain of a transcription factor such as Gal4and/or LexA. The cells additionally contain a reporter gene whosepromoter contains binding sites for the corresponding DNA-bindingdomain. By transforming the cells with a vector that expresses a secondfusion protein comprising a candidate substrate fused to an activationdomain (e.g. from Gal4 and/or herpes simplex virus VP16) expression ofthe reporter gene may be increased if the candidate substrate interactswith the infold agent. In this way, it is possible rapidly to identifynovel infold agents.

In some embodiments, any of the binding assays described herein may beperformed using a range of concentrations of infold agents and/orcandidate substrates. In some embodiments, the binding assays describedherein are used to assess the ability of a candidate substrate to bindto a infold agent over range of infold agent concentrations (e.g.greater than about 100 μg/ml, about 100 μg/ml, about 50 μg/ml, about 40μg/ml, about 30 μg/ml, about 20 μg/ml, about 10 μg/ml, about 5 μg/ml,about 4 μg/ml, about 3 μg/ml, about 2 μg/ml, about 1.75 μg/ml, about 1.5μg/ml, about 1.25 μg/ml, about 1.0 μg/ml, about 0.9 μg/ml, about 0.8μg/ml, about 0.7 μg/ml, about 0.6 μg/ml, about 0.5 μg/ml, about 0.4μg/ml, about 0.3 μg/ml, about 0.2 μg/ml, about 0.1 μg/ml, about 0.05μg/ml, about 0.01 μg/ml, and/or less than about 0.01 μg/ml).

In some embodiments, any of the binding studies described herein can beexecuted in a high throughput fashion. Using high throughput assays, itis possible to screen up to several thousand infold agents in a singleday. In some embodiments, each well of a microtiter plate can be used torun a separate assay against a selected candidate substrate, or, ifconcentration and/or incubation time effects are to be observed, every5-10 wells can test a single candidate substrate. Thus, a singlestandard microtiter plate can assay up to 96 binding interactionsbetween infold agents and candidate substrates; if 1536 well plates areused, then a single plate can assay up to 1536 binding interactionsbetween infold agents and candidate substrates; and so forth. It ispossible to assay many plates per day. For example, up to about 6,000,about 20,000, about 50,000, or more than about 100,000 assay screens canbe performed on binding interactions between infold agents and candidatesubstrates using high throughput systems in accordance with the presentinvention.

In some embodiments, such methods utilize an animal host. As usedherein, an “animal host” includes any animal model suitable forinfluenza research. For example, animal hosts suitable for the inventioncan be any mammalian hosts, including primates, ferrets, cats, dogs,cows, horses, rodents such as, mice, hamsters, rabbits, and rats. Incertain embodiments, an animal host used for the invention is a ferret.In particular, in some embodiments, an animal host is naïve to viralexposure or infection prior to administration of an infold agent(optionally in an inventive composition). In some embodiments, theanimal host is inoculated with, infected with, or otherwise exposed tovirus prior to or concurrent with administration of an infold agent. Ananimal host used in the practice of the present invention can beinoculated with, infected with, or otherwise exposed to virus by anymethod known in the art. In some embodiments, an animal host may beinoculated with, infected with, or exposed to virus intranasally.

In some embodiments, a suitable animal host may have a similardistribution of umbrella vs. cone topology glycans and/or α2,6 glycansvs. α 2,3 glycans to the distribution found in the human respiratorytract. For example, it is contemplated that a ferret as an animal hostmay be more representative than a mouse when used as model of diseasecaused by influenza viruses in humans (Tumpey, et al. Science (2007)315; 655-659). Without wishing to be bound any theories, the presentinvention encompasses the idea that ferrets may have a more similardistribution of glycans in the respiratory tract to those in the humanrespiratory tract than mouse does to human.

Naïve and/or inoculated animals may be used for any of a variety ofstudies. For example, such animal models may be used for virustransmission studies as in known in the art. It is contemplated that theuse of ferrets in virus transmission studies may serve as a reliablepredictor for virus transmission in humans. For example, airtransmission of viral influenza from inoculated animals (e.g., ferrets)to naïve animals is known in the art (Tumpey, et al. Science (2007) 315;655-659). Virus transmission studies may be used to test infold agents.For example, infold agents may be administered to a suitable animal hostbefore, during or after virus transmission studies in order to determinethe efficacy of said infold agent in blocking virus binding and/orinfectivity in the animal host. Using information gathered from virustransmission studies in an animal host, one may predict the efficacy ofan infold agent in blocking virus binding and/or infectivity in a humanhost.

Pharmaceutical Compositions and Methods of Treatment

In some embodiments, the present invention provides for pharmaceuticalcompositions including infold agents and/or related entities. Forexample, in some embodiments, infold agent polypeptides, nucleic acidsencoding such polypeptides, characteristic or biologically activefragments of such polypeptides or nucleic acids, antibodies that bind toand/or compete with such polypeptides or fragments, small molecules thatinteract with or compete with such polypeptides or with glycans thatbind to them, etc. are included in pharmaceutical compositions.

The invention encompasses treatment of influenza infection byadministration of such pharmaceutical compositions. In some embodiments,pharmaceutical compositions are administered to a subject suffering fromor susceptible to an influenza infection. In some embodiments, a subjectis considered to be suffering from an influenza infection in the subjectis displaying one or more symptoms commonly associated with influenzainfection. In some embodiments, the subject is known or believed to havebeen exposed to the influenza virus. In some embodiments, a subject isconsidered to be susceptible to an influenza infection if the subject isknown or believed to have been exposed to the influenza virus. In someembodiments, a subject is known or believed to have been exposed to theinfluenza virus if the subject has been in contact with otherindividuals known or suspected to have been infected with the influenzavirus and/or if the subject is or has been present in a location inwhich influenza infection is known or thought to be prevalent.

In some embodiments, subjects suffering from or susceptible to influenzainfection are tested for antibodies to infold agents prior to, during,or after administration of pharmaceutical compositions. In someembodiments, subjects having such antibodies are not administeredpharmaceutical compositions comprising infold agents. In someembodiments, an appropriate dose of pharmaceutical composition and/orinfold agent is selected based on detection (or lack thereof) of suchantibodies.

In some embodiments, selection of a particular subject for treatment,particular infold agent or composition for administration, and/orparticular dose or regimen for administration, is memorialized, forexample in a written, printed, or electronic storage form.

Inventive compositions may be administered prior to or after developmentof one or more symptoms of influenza infection.

The invention encompasses treatment of influenza infections byadministration of agents described herein.

The present invention also provides other therapeutic compositionsuseful in the treatment of viral infections. In some embodiments,treatment is accomplished by administration of an agent that interfereswith expression or activity of an HA polypeptide.

In some embodiments, the present invention provides pharmaceuticalcompositions comprising antibodies or other agents related to providedinfold agents. For example, the invention provides compositionscontaining antibodies that recognize infold agents, nucleic acids (suchas nucleic acid sequences complementary to sequences of infold agents,which can be used for RNAi), or combination thereof. In someembodiments, collections of different agents, having diverse structuresare utilized. In some embodiments, therapeutic compositions comprise oneor more multivalent agents. In some embodiments, treatment comprisesurgent administration shortly after exposure or suspicion of exposure toinfluenza virus.

In general, a pharmaceutical composition will include a therapeuticagent in addition to one or more inactive agents such as a sterile,biocompatible carrier including, but not limited to, sterile water,saline, buffered saline, or dextrose solution. Alternatively oradditionally, the composition can contain any of a variety of additives,such as stabilizers, buffers, excipients (e.g., sugars, amino acids,etc), or preservatives.

In certain embodiments, the therapeutic agent present in an inventivepharmaceutical composition will consist of one or more infold agents asdescribed herein. In some embodiments, an inventive pharmaceuticalcomposition contains an infold agent that binds to HA polypeptides orumbrella topology glycans (and/or to umbrella topology glycan mimics).In some such embodiments, the inventive composition is substantiallyfree of related agents (e.g., of other infold agents, etc.) that do notbind to umbrella-topology glycans. In some such embodiments, thepharmaceutical compositions contains not more than least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90% or more of an agent that binds to a glycosylated or non-glycosylatedHA polypeptide and/or HA receptor glycans other than umbrella topologyglycans.

In certain embodiments, a pharmaceutical composition will include atherapeutic agent that is encapsulated, trapped, or bound within a lipidvesicle, a bioavailable and/or biocompatible and/or biodegradablematrix, or other microparticle.

In some embodiments, a provided pharmaceutical composition will includean infold agent that is not aggregated. For example, in someembodiments, less than 1%, 2%, 5%, 10%, 20%, or 30%, by dry weight ornumber, of infold agents is present in an aggregate.

In some embodiments, a provided pharmaceutical composition will includean infold agent that is not denatured. For example, in some embodiments,less than 1%, 2%, 5%, 10%, 20%, or 30%, by dry weight or number, ofinfold agents administered is denatured.

In some embodiments, a provided pharmaceutical composition will includean infold agent that is not inactive. For example, in some embodiments,less than 1%, 2%, 5%, 10%, 20%, or 30%, by dry weight or number, ofinfold agents administered is inactive.

In some embodiments, pharmaceutical compositions are formulated toreduce immunogenicity of provided infold agents. For example, in someembodiments, a provided infold agent is associated with (e.g., bound to)an agent, such as polyethylene glycol and/or carboxymethyl cellulose,that masks its immunogenicity. In some embodiments, a provided bindingagent has additional glycosylation that reduces immunogenicity.

Combination Therapy

Pharmaceutical compositions of the present invention may be administeredeither alone or in combination with one or more other therapeutic agentsincluding, but not limited to, vaccines and/or antibodies. By “incombination with,” it is not intended to imply that the agents must beadministered at the same time or formulated for delivery together,although these methods of delivery are within the scope of theinvention. In general, each agent will be administered at a dose and ona time schedule determined for that agent. Additionally, the inventionencompasses the delivery of the pharmaceutical compositions incombination with agents that may improve their bioavailability, reduceor modify their metabolism, inhibit their excretion, or modify theirdistribution within the body. Although the pharmaceutical compositionsof the present invention can be used for treatment of any subject (e.g.,any animal) in need thereof, they are most preferably used in thetreatment of humans.

In some embodiments, pharmaceutical compositions of the presentinvention and/or one or more infold agents are administered incombination with one or more anti-viral agents. In some embodiments,such anti-viral agents include, but are not limited to, acyclovir,ribavirin, amantadine, remantidine, zanamivir (RELENZA®), oseltamivir(TAMIFLU®), amantadine, rimantadine and/or combinations thereof.

In some embodiments, pharmaceutical compositions of the presentinvention and/or one or more infold agents are administered incombination with one or more antibodies. In some embodiments, theantibodies bind HA polypeptides on the virus. In some embodiments, theantibodies bind the MPER region of the HA polypeptide (e.g., H1, H2, H3,H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16polypeptides). In some embodiments, the antibodies bind a glycosylatedMPER region of the HA polypeptide. In some embodiments, the antibodiesbind the HA receptor. In some embodiments, the antibodies bind tosialyated glycans on the HA receptor. In some embodiments, theantibodies are C179, F10 and CR6261.

In some embodiments, pharmaceutical compositions of the presentinvention and/or one or more infold agents are administered incombination with one or more anti-viral agents. In some embodiments,such anti-viral agents include, but are not limited to, acyclovir,ribavirin, amantadine, remantidine, zanamivir (Relenza), oseltamivir(Tamiflu), amantadine, rimantadine and/or combinations thereof.

In some embodiments, pharmaceutical compositions of the presentinvention and/or one or more infold agents are administered incombination one or more vaccines. In some embodiments, the vaccine is aanti-viral vaccine. In some embodiments, the vaccine is ananti-influenza vaccine. In some embodiments, the anti-influenza vaccineis to treat seasonal influenza (e.g., commonly referred to as the flu).In some embodiments, the anti-influenza vaccine is the flu shot and/orFluMist. In some embodiments, the anti-influenza vaccine is targeted toa specific combination of one or more HA polypeptides (e.g., H1, H2, H3,H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16polypeptides). In some embodiments, the anti-influenza vaccine isspecific for one or more combinations of H1N1, H2N2, H3N2, H5N1, H7N7,H1N2, H9N2, H7N2, H7N3, or H10N7 viruses. In some embodiments, theanti-influenza vaccine is specific to H1N1 viruses. In some embodiments,the anti-influenza vaccine is specific to H3N2 viruses. In someembodiments, the anti-influenza vaccine is specific to H1N1 and H3N2viruses.

In some embodiments pharmaceutical compositions and/or one or moreinfold agents may be administered in combination with one or more otherpharmaceutical agents used to treat the symptoms associated withinfluenza virus infection. In some embodiments, pharmaceutical agentsused to treat the symptoms associated with influenza infection are painrelievers, anti-inflammatories, antibiotics and/or combinations thereof.In some embodiments, pharmaceutical agents used to treat the symptomsassociated with influenza infection are acetaminophen, ibuprofen,aspirin, naproxen and/or combinations thereof.

Methods of Administration

Pharmaceutical compositions of the present invention can be administeredby a variety of routes, including oral, intravenous, intramuscular,intra-arterial, subcutaneous, intraventricular, transdermal,interdermal, rectal, intravaginal, intraperitoneal, topical (as bypowders, ointments, creams, or drops), mucosal, nasal, buccal, enteral,sublingual; by intratracheal instillation, bronchial instillation,and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.In general the most appropriate route of administration will depend upona variety of factors including the nature of the agent (e.g., itsstability in the environment of the gastrointestinal tract), thecondition of the patient (e.g., whether the patient is able to tolerateoral administration), etc.

At present the oral or nasal spray or aerosol route (e.g., byinhalation) is most commonly used to deliver therapeutic agents directlyto the lungs and respiratory system. However, the invention encompassesthe delivery of the inventive pharmaceutical composition by anyappropriate route taking into consideration likely advances in thesciences of drug delivery.

In some embodiments, preparations for inhaled or aerosol deliverycomprise a plurality of particles. In some embodiments, suchpreparations have a mean particle size of 4, 5, 6, 7, 8, 9, 10, 11, 12,or 13 microns. In some embodiments, preparations for inhaled or aerosoldelivery are formulated as a dry powder. In some embodiments,preparations for inhaled or aerosol delivery are formulated as a wetpowder, for example through inclusion of a wetting agent. in someembodiments, the wetting agent is selected from the group consisting ofwater, saline, or other liquid of physiological pH.

In some embodiments, inventive compositions are administered as drops tothe nasal or buccal cavity. In some embodiments, a dose may comprise aplurality of drops (e.g., 1-100, 1-50, 1-20, 1-10, 1-5, etc.)

In some embodiments, inventive compositions are administered using adevice that delivers a metered dosage of composition (e.g., of infoldagent).

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. No. 4,886,499, U.S. Pat. No. 5,190,521, U.S. Pat.No. 5,328,483, U.S. Pat. No. 5,527,288, U.S. Pat. No. 4,270,537, U.S.Pat. No. 5,015,235, U.S. Pat. No. 5,141,496, U.S. Pat. No. 5,417,662.Intradermal compositions may also be administered by devices which limitthe effective penetration length of a needle into the skin, such asthose described in WO99/34850, incorporated herein by reference, andfunctional equivalents thereof. Also suitable are jet injection deviceswhich deliver liquid compositions to the dermis via a liquid jetinjector or via a needle which pierces the stratum corneum and producesa jet which reaches the dermis. Jet injection devices are described forexample in U.S. Pat. No. 5,480,381, U.S. Pat. No. 5,599,302, U.S. Pat.No. 5,334,144, U.S. Pat. No. 5,993,412, U.S. Pat. No. 5,649,912, U.S.Pat. No. 5,569,189, U.S. Pat. No. 5,704,911, U.S. Pat. No. 5,383,851,U.S. Pat. No. 5,893,397, U.S. Pat. No. 5,466,220, U.S. Pat. No.5,339,163, U.S. Pat. No. 5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat.No. 5,064,413, U.S. Pat. No. 5,520,639, U.S. Pat. No. 4,596,556, U.S.Pat. No. 4,790,824, U.S. Pat. No. 4,941,880, U.S. Pat. No. 4,940,460, WO97/37705 and WO 97/13537. Also suitable are ballistic powder/particledelivery devices which use compressed gas to accelerate compositions inpowder form through the outer layers of the skin to the dermis.Additionally, conventional syringes may be used in the classical mantouxmethod of intradermal administration.

Formulations

General considerations in the formulation and manufacture ofpharmaceutical agents may be found, for example, in Remington'sPharmaceutical Sciences, 19^(th) ed., Mack Publishing Co., Easton, Pa.,1995.

Pharmaceutical compositions may be administered in any dose appropriateto achieve a desired outcome. In some embodiments, the desired outcomeis reduction in intensity, severity, and/or frequency, and/or delay ofonset of one or more symptoms of influenza infection.

In some embodiments, pharmaceutical compositions are formulated toadminister a dose of infold agent effective to compete with an influenzaHA polypeptide for binding to umbrella topology glycans. In someembodiments, such binding by an influenza HA polypeptide is reducedafter administration of one or more doses of an infold agent compositionas compared with its level absent such administration. In someembodiments, pharmaceutical compositions are formulated to administer adose of infold agent effective to saturate at least 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more HA polypeptide bindingsites (e.g., HA polypeptide binding sites containing umbrella topologyglycans) present in the subject (e.g., in the respiratory tract of thesubject) receiving the composition.

In some embodiments, pharmaceutical compositions are formulated todeliver a unit dose of infold agent within the range of 0.0001 to 1000nmg/kg.

In some embodiments, pharmaceutical compositions are administered inmultiple doses. In some embodiments, pharmaceutical compositions areadministered in multiple doses/day. In some embodiments, pharmaceuticalcompositions are administered according to a continuous dosing regimen,such that the subject does not undergo periods of less than therapeuticdosing interposed between periods of therapeutic dosing. In someembodiments, pharmaceutical compositions are administered according toan intermittent dosing regimen, such that the subject undergoes at leastone period of less than therapeutic dosing interposed between twoperiods of therapeutic dosing.

Diagnostics/Kits

In some embodiments, the present invention provides kits for detectinginfold agents as described herein whether or not such polypeptides areinfold agents.

In some embodiments, the present invention provides kits for detectinginfold agents and particular for detecting infold agents with particularHA polypeptide and/or glycan binding characteristics (e.g., binding toumbrella glycans, to α2,6 sialylated glycans, to long α2,6 sialylatedglycans, etc.) in pathological samples, including, but not limited to,blood, serum/plasma, peripheral blood mononuclear cells/peripheral bloodlymphocytes (PBMC/PBL), sputum, urine, feces, throat swabs, dermallesion swabs, cerebrospinal fluids, cervical smears, pus samples, foodmatrices, and tissues from various parts of the body such as brain,spleen, and liver. The present invention also provides kits fordetecting infold agents of interest in environmental samples, including,but not limited to, soil, water, and flora. Other samples that have notbeen listed may also be applicable.

In some embodiments, methods for detecting infold agents involveproviding a pathological and/or environmental sample, contacting thesample with an infold agent, and determining whether the infold agentbinds to the sample relative to a negative control binding agent. Insome embodiments, such methods involve a step of processing the sample(e.g., subjecting the sample to one or more purification steps) prior tothe step of contacting. In some embodiments, provided infold agents arelabeled with a detectable moiety (e.g., fluorescent, radioactive,chemoluminescent label, etc.). In some embodiments, infold agents aredetectable via immunological methods (e.g., western blotting, ELISA,immunofluorescence, etc.). In some embodiments, infold agents areimmobilized (e.g., to a bead, to a microtiter dish, to an array, to aglycan array, etc.) prior to the step of contacting.

In certain embodiments, inventive kits may include one or more agentsthat specifically detect infold agents with HA polypeptide and/orparticular glycan binding characteristics. Such detecting agents mayinclude, for example, antibodies that specifically recognize certaininfold agents (e.g., infold agents that bind to umbrella glycans and/orto α2,6 sialylated glycans and/or to long α2,6 sialylated glycans),which can be used to specifically detect such infold agents by ELISA,immunofluorescence, and/or immunoblotting.

Antibodies that bind to infold agents can also be used in virusneutralization tests, in which a sample is treated with antibodyspecific to infold agent of interest, and tested for its ability toinfect cultured cells relative to untreated sample. If the virus in thatsample contains such infold agents, the antibody will neutralize thevirus and prevent it from infecting the cultured cells. Alternatively oradditionally, such antibodies can also be used in HA-inhibition tests,in which the HA protein is isolated from a given sample, treated withantibody specific to a particular infold agents or set of infold agents,and tested for its ability to agglutinate erythrocytes relative tountreated sample. If the virus in the sample contains such an infoldagent, the antibody will neutralize the activity of infold agents andprevent it from agglutinating erythrocytes (Harlow & Lane, Antibodies: ALaboratory Manual, CSHL Press, 1988;who.int/csr/resources/publications/influenza/WHO_CDS_CSR_NCS_2002_5/en/index;who.int/csr/disease/avian_influenza/guidelines/labtests/en/index). Insome embodiments, such agents may include nucleic acids thatspecifically bind to nucleotides that encode particular infold agentsand that can be used to specifically detect such infold agents by RT-PCRor in situ hybridization(who.int/csr/resources/publications/influenza/WHO_CDS_CSR_NCS_2002_5/en/index;who.int/csr/disease/avian_influenza/guidelines/labtests/en/index). Incertain embodiments, nucleic acids which have been isolated from asample are amplified prior to detection. In certain embodiments,diagnostic reagents can be detectably labeled.

The present invention also provides kits containing reagents accordingto the invention for the treatment of influenza virus infection.Contents of the kits include, but are not limited to, expressionplasmids containing infold agent nucleotides (or characteristic orbiologically active portions) encoding infold agents of interest (orcharacteristic or biologically active portions). Alternatively oradditionally, kits may contain expression plasmids that express infoldagents of interest (or characteristic or biologically active portions).Expression plasmids containing no virus genes may also be included sothat users are capable of incorporating infold agent nucleotides fromany influenza virus of interest. Mammalian cell lines may also beincluded with the kits, including but not limited to, Vero and MDCK celllines. In certain embodiments, diagnostic reagents can be detectablylabeled.

In certain embodiments, kits for use in accordance with the presentinvention may include, a reference sample, instructions for processingsamples, performing the test, instructions for interpreting the results,buffers and/or other reagents necessary for performing the test. Incertain embodiments the kit can comprise a panel of antibodies.

The present invention provides kits for administration of pharmaceuticalcompositions. For example, in some embodiments, the invention provides akit comprising at least one dose of an infold agent. In someembodiments, the invention provides a kit comprising an initial unitdose and one or more subsequent unit doses of an infold agent. In somesuch embodiments, the initial unit dose is greater than the subsequentunit doses or wherein the all of the doses are equal.

In some embodiments, inventive kits (particularly those foradministration of infold agent pharmaceutical compositions) comprise atleast one component of a delivery device, e.g., an inhaler. In some suchembodiments, the invention provides a kit comprising at least onecomponent of a delivery device, e.g., an inhaler and a dose of an of aninfold agent.

In some embodiments, provided kits comprise instructions for use.

EXEMPLIFICATION Example 1 Design of Infold Agents

The present example illustrates the design of infold agents to bind tospecific regions on an HA polypeptide.

One exemplary infold sequence was designed against the HA bindinginterface (FIG. 6). These interactions revealed several potentiallystabilizing contacts with both the head (HA-1) and stalk (HA-2) domainsof HA (Table 10). It is seen that one of the regions of the designedprotein has the desired stabilizing hydrophobic contacts and hydrogenbonds with both HA-1 and HA-2 domains, while another prominent regionhas stabilizing hydrophobic interactions primarily with HA-1, while yetanother region has stabilizing hydrophobic interactions primarily withHA-2. Thus, the designed infold protein under consideration is expectedto bind at the stalk-head interface of the HA-1 and HA-2 domains.Furthermore, as expected, the designed proteins are seen to accommodatethe N-glycosylation in the HA MPER region owing to their significantlysmaller volume than antibodies (FIG. 6). Thus, supporting that thedesigned proteins will bind with good affinity to both MPER-glycosylatedand MPER-non-glycosylated influenza strains, supporting universalinfluenza neutralization.

TABLE 10 Structural characterization of Infold-2 designed protein-HAbinding interface Stalk-protein (HA-2-Infold) Interface Head-protein(HA-1-Infold) Interface Number of interface residues (15:11) Number ofinterface residues (10:9) Number of interface atoms (47:43) Number ofinterface atoms (34:27) Solvent accessible interface area Solventaccessible interface area (411.0 A²:435.3 A²) (241.7 A²:239.3 A²) Numberof Hydrogen bonds: 4 Number of Hydrogen bonds: 3 Gln42_OE1 - Gly78_NGln34_NE2 - Thr22_O Gln42_OE1 - Gly76_N Ser292_O - Thr22_OG1 Asp19_O -Tyr75_OH Gln34_OE1 - Thr23_OG1 Thr49_OG1 - Arg26_NH2 Important interfaceresidues: Important interface residues: Stalk: Val18, Asp19, Gly20,Trp21, Lys38, Head: His12, Asn13, Ser16, Glu18, Thr31, Thr41, Gln42,Ile45, Asp46, Val48, Thr49, His32, Gln34, Ser292, Met293, Thr319 Asn50,Val52, Asn53, Ile56 Infold: Glu18, Val21, Thr22, Thr23, Ser24, Infold:Val21, Thr23, Ser25, Arg26, Phe49, Ser25, Arg26, Phe49, Met50 Val74,Tyr75, Gly76, Arg77, Gly78, Asp79

Targeting the delivery of therapeutics locally instead of globally isknown to provide immensely less-toxic treatment with higher potency. Inaddition to the broad spectrum influenza neutralization properties, theability to specifically bind α2,6 sialylated glycans on the HA receptorbecomes an important property of the novel therapeutics for influenza.Without wishing to be bound by any theories, we propose the followingstrategies for targeted broad spectrum influenza neutralization asillustrated in FIG. 1. They include the ability to bind to theglycosylated/non-glycosylated conserved membrane proximal epitope regionof the HA (broad spectrum neutralization) along with the ability to bindto sialylated glycan receptors in the human upper respiratory tract(targeted delivery).

Example 2 Infold Agents Bind HA Polypeptides

The present example illustrates binding of infold agents to HApolypeptide in an in vitro binding assay.

Maxisorp 96-well plate wells were coated with 0.2 μg an HA polypeptideof different subtypes (H1, H3, H5, H7 and H9) and left overnight at 4°C. The HA polypeptide coated plates were washed thrice with PBS andblocked with 1% BSA in PBST. Different concentrations of infold agentsalong with C179 antibody (control) were added to HA polypeptide coatedwells and the plate was incubated at RT for 2 hrs. The plate was washedthrice with PBST and the wells containing infold agents were incubatedwith mouse-anti-6×His antibody (1:1000 dilution) for 1 hr at RT. Theplates were washed thrice with PBST and all wells were incubated withgoat-anti-mouse HRP antibody for 1 hr at RT. Post-incubation the wellswere washes with PBST and the bound HRP was measured using TMBsubstrate. TMB substrate was added to the wells, incubated for 3minutes, followed by addition of 1 N sulphuric acid. Absorbance wasmeasured at 450 nm.

Our experimental results show good agreement with the predictedtheoretical calculations with the designed proteins displaying highaffinity towards both MPER-glycosylated and MPER-non-glycosylatedstrains of influenza. Our results show that an exemplary infold agentbinds to various glycosylated and non-glycosylated HA polypeptides (H1,H3, H5, H7 and H9) with similar affinities (FIG. 7, left panel). Thesedata are in comparison to the C179 antibody control (FIG. 7, rightpanel), which shows that the C179 antibody cannot distinguish betweendifferent HA polypeptide clades.

Example 3 Binding Affinity Between Infold Agents and the Targets of anInfold Agent

The present example shows a calculation of binding affinity, asrepresented as a dissociation constant (K_(d)), between an infold agentand the target of the infold agent. In this example, the infold agent isan exemplary infold agent and the target of the infold agent is an HApolypeptide.

Binding affinity between the exemplary infold agent and an HApolypeptide is a function of the concentrations of both the infold agentand the HA polypeptide. In the present example, the binding affinity isquantitatively described using dissociation constant (K_(d)). An exampleof how to measure the dissociation constant is described below.

HA polypeptide coated plates were used to perform ELISA assays with anexemplary infold agent as described previously. The measured absorbanceat 450 nm was used to calculate the fractional saturation of thereceptor. The fractional saturation was plotted as a function of molarconcentration of the infold agent. The data was fit to the followingequation:

$y = \frac{I_{0}}{( {K_{d} + I_{0}} )}$

where y is the fractional saturation, I₀ is the concentration of theinfold agent and K_(d) is the dissociation constant.

Using the above referenced calculation, and applying regressionanalysis, we have observed K_(d) values in the range of 0.1 to 500 nMfor binding of infold agents to HA polypeptides. In some embodiments, wehave observed K_(d) values in the range of 10 to 100 nM for binding ofinfold agents to HA polypeptides. In some embodiments, we have observedK_(d) values in the range of 50 to 100 nM for binding of infold agentsto HA polypeptides.

Example 4 Infold Agents Inhibit Virus Infectivity In Vitro

The present example illustrates the ability of infold agents to preventvirus infectivity in in vitro binding assays.

The ability of an infold agent to inhibit influenza infection wasevaluated in vitro using MDCK (Madin-Darby Canine Kidney) cells, anepithelial cell line commonly used for the propagation and testing ofinfluenza virus strains. The inhibitory effects of the infold agent oninfectivity were determined by measuring both viral yield and the extentof influenza-induced cytopathic effects (CPE) on the host cells. Whileplaque assay (FIGS. 15 and 16) and qRT-PCR were employed to quantifyviral production, and a cell viability assay was used to measure CPElevels. The experiments were set up to allow for the infold agent tofirst bind to its viral target during a one hour pre-incubation periodbefore introduction to the host cells. Infection was carried out in thepresence of low levels of trypsin (1 μM). The plaque assay was performedby inoculating confluent monolayer of cells with serial dilutions oftest samples and overlaying with a viscous suspension of the polymerAvicel (FMC Biopolymers). Plaques were allowed to develop over a periodof 48 hours@ 35° C., formalin fixed, stained with crystal violet andvisualized (FIGS. 15 and 16). The plaque count was used to calculateinfectious viral titers in the test samples. Total viral output was alsodetermined by quantitative RT-PCR, which measured levels of viral genomecopies in the infected samples. The primers and labeled probe weredesigned to specifically amplify and measure a region within the viralhemagglutinin gene by the TaqMan method. The relative number of viablecells following infection was used as a measure of CPE. Sub-confluentcell cultures were exposed to a compound/virus mix (moi=1.0) for aperiod of one hour@ 35° C. Unbound virus and drug were then removed andreplaced with virus growth medium. Cell viability was determinedfollowing 48 hours incubation using Promega's CellTiter Blue reagents(resazurin) as the extent of the metabolic conversion of thenon-fluorescent resazurin to fluorescent resorufin read (555/585 nmexcitation/emission; SpectraMax M2; Molecular Probes).

From these studied, we have found that infold agents inhibitvirus-induced plaque production. In some embodiments, infold agentsinhibit virus-induced plaque production in a dose-dependent manner.

Example 5 Infold Agents Bind HA Polypeptides In Vivo

The present example illustrates the ability of infold agents to bind HApolypeptides in in vivo.

BALB/c mice (4-6 weeks old) were procured from Charles River Labs. Themice were weighed and divided into four groups of 6 mice each for theexperiment. Each group was administered with isoflurane, dose with 0mg/kg, 0.06 mg/kg, 0.6 mg/kg or 6 mg/kg of infold agent and allowed torecover (<2 min). They were then re-administered with isoflurane andchallenged intranasally with a lethal dose of H1N1 PR8 virus. The micewere monitored daily for 14 days. The weight loss, visual score, andsurvival were recorded daily (FIGS. 17 and 18). Clinical signs ofinfluenza infection in mice include hunched posture, ruffled fur, rapidbreathing, loss of appetite, weight loss, and death. In addition, nasalwashes were collected on day 3 from three animals each from theuntreated group and group dosed with 6 mg/kg in order to demonstrate areduction in viral replication in the nose with peak titers expected atday 3. The lungs were harvested from each mouse prior to sacrifice.

The results of these studies have shown that infold agents cansuccessfully delay the onset of H1N1 infection in mice, with resultsthat were comparable, or exceedingly better than an alternativeanti-viral treatment, Ribavirin.

Example 6 Infold Agents in Diagnostics

The present example illustrates the ability of infold agents to providea rapid way for (a) identifying the presence of influenza virus in abiological sample and (b) characterizing the virus, based on thesubtype.

A sandwich ELISA (virus typing ELISA assay) assay is used for thepurpose of identifying the presence of influenza virus andcharacterizing the virus subtype. For the virus typing ELISA assay,96-well plates will be coated with 2 μg of infold agent and incubatedovernight at 4° C. The plates would then be extensively washed with PBSand blocked with 1% BSA in PBST for 1 hr. Post blocking, the plates willbe washed with PBST and stored at 4° C. till further use.

Biological samples suspected of containing influenza virus will bediluted in sample buffer (PBS) either directly or post processing. Thediluted samples will then be applied to the infold-coated wells andincubated for 2 hrs at room temperature (RT) followed by extensivewashing. Virus from the sample would thus be captured by the infold andlend itself for further analysis. Subtype specific antibody will beapplied to different wells for 1 hr at RT. After further washes withPBST, HRP-conjugated secondary antibodies will be applied to the wells.Post-incubation, the wells would be washed, and treated with TMBsubstrate and 1 N sulphuric acid. The absorbance at 450 nm will bemeasured using a spectrophotometer. Appropriate negative and positivecontrols will be included.

The results of the virus typing ELISA assay will yield information aboutthe presence of influenza virus in a sample and the subtype of thevirus.

Example 7 Infold Agents for Influenza Virus Glycan Characterization

The present example illustrates the ability of infold agents as means toenrich and label influenza virus for glycan characterization using aglycan typing assay.

In the glycan typing assay, the infold agents would be conjugate to Qdot525 Carboxyl Quantum Dots using EDC chemistry as per manufacturersinstruction. The Qdot-infold complex will be added to processedbiological samples and stirred well for 2 hrs. The sample will then becentrifuged and the Qdot-infold-InfA complex would be washed thrice withPBST. This complex would then be applied on glycan array (containingumbrella and cone topology glycans). After incubating for 2 hrs at RT,the wells would be washed thrice with PBST. The bound fluorescence wouldbe measured using SpectraMax M2e spectrophotometer using bottom readmode.

The results of this assay will be yield information regarding the glycancharacterization of influenza viruses.

Example 8 IC₅₀ Evaluation of Infold-28

The IC₅₀ of anti-influenza infold agents targeting HA have beenquantified. These studies utilized the H1N1 influenza strain PR8(A/Puerto Rico/8/34). In brief, confluent MDCK cells are infected withPR8 [4E3 PFU/mL] pre-incubated for 40 minutes with varyingconcentrations of anti-influenza agents. After one hour of infection,media was removed and replaced with virus-free, drug-containing media orvirus-free, drug-free media depending upon the experiment. After 48hours of incubation at 37° C., 5% CO₂, supernatants were collected.Viral RNA was isolated, and viral titer was quantified by real time PCRusing primers specific for the virus M protein.

Initial assessment of the IC₅₀ values for Infold-28 were determined bymicroneutralization assay followed by quantitative PCR (qPCR). Mixturesof virus (PR8) and Infold-28 at various titers and concentrations,respectively, were pre-incubated for 1 hour at 35° C. before beingapplied to MDCK cell cultures in a 96-well tissue culture plate (˜10,000cells/well). After additional 48 hours of incubation, the culture mediumwas collected from each well for viral yield determination by qPCR. Apreliminary indication of Infold-28 neutralizing activity came fromstaining of cells that survived infection with crystal violet (FIG. 20;stained cells are in black) which showed drug concentration-dependentand viral titer-dependent inhibition.

Media from triplicate samples were combined and then subjected to directquantification of viral yield by qPCR. Viral titers were calculated fromthe PCR Ct values with the aid of an internal standard curve, and theIC₅₀ values were determined by plotting the calculated titers againstInfold-28 concentrations (FIG. 21). The results showed 50% inhibition(IC₅₀) of 200 pfu/ml (moi=0.04) of PR8 viral particles with 11 μg/mlInfold-28. As expected for an active agent, this value increased withviral titer. Results show that Infold-28 is a potent inhibitor; IC₅₀ isinfluenced by the multiplicity of infection (moi).

We also investigated how the method of addition of Infold-28 (i.e., inthe overlay, etc.) influenced inhibition. The IC₅₀ of Infold-28 wasmeasured to be ˜1 μg/mL or ˜6 nM when drug is added to the overlay afterinfection, and ˜8 μg/mL or ˜50 nM when drug is not added to the overlayafter infection (FIG. 22). At least one other tested infold agent fromTable 9 showed a comparable IC₅₀, whereas less potent activity wasobserved with at least one other such agent. Those of ordinary skill inthe art will appreciate that the guidance provided herein permitsadjustment and optimization of infold agents based on the representativeagents provided in Table 9 without undue experimentation.

Example 9 Minimum Inhibitory Activity Assay

We have utilized a method to determine the minimum inhibitoryconcentration (MIC) of the antiviral agents against influenza A. To beactive in this assay, the agent must bind to the virus and neutralizethe virus' ability to form plaques. Briefly, an agent is seriallydiluted in two fold increments in PBS to form a concentration gradientacross multiple wells. A known number of viral plaque forming units areadded to each well and after 1 hour incubation, the mixture is added toan MDCK monolayer to allow viral binding. An Avicel overlay encouragesplaque formation and the plaques are visualized by immunostain. Thelowest concentration of agent to prevent plaque formation is reported asthe MIC. These studies utilized H1N1 strain PR8 (A/Puerto Rico/8/34).

Representative infold agents, including Infold-28, from Table 9 showedactivity in the assay versus H1N1, and specifically showed MIC<120, andeven within a range of 15 to about 100, or about 15 to about 60, orabout 15-20 to about 60-100. Infold agents tested in this assay did notshow activity versus an H3N2 virus in the particular studies performed.At least in some cases where no activity was observed (whether versusH1N1, H3N2, or both) may have been issues with stability of infold agentin the assay. Those of ordinary skill in the art will appreciate thatimprovements to the stability of such agents (e.g., via modification ofamino acid sequence or backbone), steady or repeat infusion of agent, orother experimental adjustments to agents and/or conditions of the assaymay reveal activity not seen in the particular test thusfar performed.

Example 10 PEGylation of Infold-28

Various PEG were added to Infold-28 and MIC was determined. We observeda trend where PEGylation with larger PEG molecules (e.g., 20 kD)negatively impacts Infold-28's performance in MIC assays, while smallerPEG molecules (e.g., 5 kD) appear to be better tolerated.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims:

1-65. (canceled)
 66. A method of making a pharmaceutical compositioncomprising the step of: formulating an infold agent with one or moreinactive agents or additives, wherein the infold agent comprises aninfold selected from the group consisting of Infold-1, Infold-2,Infold-3, Infold-4, Infold-5, Infold-6, Infold-7, Infold-8, Infold-9,Infold-10, Infold-11, Infold-12, Infold-13, Infold-14, Infold-15,Infold-16, Infold-17, Infold-18, Infold-19, Infold-20, Infold-21,Infold-22, Infold-23, Infold-24, Infold-25, Infold-26, Infold-27,Infold-28, Infold-29, Infold-30, Infold-31, Infold-32, Infold-33,Infold-34, Infold-35, Infold-36, Infold-37, Infold-38, Infold-39, andInfold-40 to provide a pharmaceutical composition comprising at leastone unit dose of the infold agent.
 67. The method of claim 66, whereinthe inactive agent is a sterile biocompatible carrier.
 68. The method ofclaim 67, where the sterile biocompatible carrier is selected from oneor more of sterile water, saline, buffered saline, and dextrosesolution.
 69. The method of claim 66, wherein the additive is selectedfrom one or more of stabilizer, excipient, and preservatives.
 70. Themethod of claim 69, wherein the excipient is selected from one or moreof sugars and amino acids.
 71. The method of claim 66, wherein thepharmaceutical composition is formulated for administration by a routeselected from the group consisting of oral, intravenous, intramuscular,intra-arterial, subcutaneous, intraventricular, transdermal,interdermal, rectal, intravaginal, intraperitoneal, topical, mucosal,nasal, buccal, enteral, sublingual, intratracheal or bronchial.
 72. Themethod of claim 71, wherein the administration is by intratracheal orbronchial instillation.
 73. The method of claim 71, wherein theadministration is by inhalation.
 74. The method of claim 66, wherein thepharmaceutical composition is formulated as an oral spray, a nasalspray, or an aerosol.
 75. The method of claim 71, wherein theadministration is by oral.
 76. The method of claim 66, wherein thepharmaceutical composition is or comprises an oral spray, a nasal sprayor an aerosol.